Methods for preventing toxicity of platinum drugs

ABSTRACT

Methods for reducing platinum drug-induced toxicity in a cell expressing Organic Cation Transporter 2 (OCT2), methods for reducing platinum drug-induced toxicity in a subject, methods for treating cancer, methods for increasing efficacy of platinum drug treatment in a subject, and pharmaceutical compositions are described. The described methods and compositions include an OCT2 inhibitor where the OCT2 inhibitor is buflomedil or a salt thereof, dolutegravir or a salt thereof, contains an imidazole, or is miconazole or a salt thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/744,485, which adopts the international filing date of Jul. 15, 2016,which is a U.S. national phase application under 35 U.S.C. § 371 ofInternational Application No. PCT/US2016/042673, filed on Jul. 15, 2016,which claims the priority benefit of U.S. Provisional Application No.62/193,529, filed on Jul. 16, 2015, the disclosures of each of which arehereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to compounds that are able to reducetoxicity of platinum drugs.

BACKGROUND OF THE INVENTION

Platinum drugs, such as oxaliplatin and cisplatin and carboplatin arewidely used highly effective chemotherapeutic drugs for treating avariety of cancers.

However, platinum drugs cause serious side effects, which deterioratepatients' quality of life, lead to early treatment termination, causesevere impairment and death. Besides hematotoxicity, nephrotoxicity,peripheral neuropathy and ototoxicity are three most prevalenttoxicities associated with platinum drugs.

Nephrotoxicity is frequently seen in patients treated with cisplatin. Itis estimated that 28%-36% of human patients receiving an initial dose of50-100 mg/m² cisplatin develop acute kidney failure; most patients whodevelop some degree of cisplatin-induced kidney injury never fullyrecover [Barabas 2008]. In contrast, nephrotoxicity is rarely seen inpatients treated with oxaliplatin and carboplatin. The clinicalcharacteristics, mechanisms and management (including diagnosis,prevention and treatment) of platinum-induced nephrotoxicity aremeticulously reviewed in [Barabas 2008, Yao 2007, Miller 2010, Karasawa2015].

Ototoxicity is frequently associated with cisplatin and carboplatin, butrarely seen in oxaliplatin-treated patients. The incidence of overallcisplatin induced ototoxicity ranges from 42% to 62% [Langer 2013]. Theclinical characteristics, mechanisms and management (includingdiagnosis, prevention and treatment) of platinum-induced ototoxicity aremeticulously reviewed in [Langer 2013, Barabas 2008, Karasawa 2015].

Peripheral neuropathy (PN) or peripheral neurotoxicity is a very commonadverse effect associated with many chemotherapeutics, includingoxaliplatin and cisplatin. Peripheral neuropathies caused by differentchemotherapeutic drugs often have distinct pathophysiology and clinicalcharacteristics [Miltenburg 2014, Hershman 2014, Wolfgang 2014]. Forexample, unlike vincristine, which primarily causes axonal damage, thedorsal root ganglion (DRG) appears to be the primary site of neuraldamage caused by cisplatin and oxaliplatin [Miltenburg 2014, Holmes1998]. Cisplatin PN is related to the cumulative dose and doseintensity. Most patients completing a full course of cisplatin treatment(usual cumulative dose 300-450 mg/m²) develop moderate-to-severe (Grade2 or higher) PN, with debilitating symptoms such as severe paresthesiasand dysesthesias, sensory loss and sensory ataxia, which can last frommonths to years after treatment is discontinued. In contrast tocisplatin PN, oxaliplatin-induced PN (OXAIPN) has both acute and chronicforms. The chronic form has pathogenesis and clinical characteristicssimilar to cisplatin PN. Severe PN (Grade 3 or higher) has been observedwith cumulative doses of oxaliplatin ranging from 510-765 mg/m² in up to10% of patients. It is reported that approximately 50% of patientsreceiving cumulative doses of oxaliplatin above 1000 mg/m² developsevere PN [Argriou 2012]. Acute OXAIPN, which is a unique, acute,transient peripheral nerve hyperexcitability syndrome, is experienced bynearly all (up to 90%) patients. Distal and perioral cold-inducedparesthesias and dysesthesias are the most commonly reported symptoms ofacute OXAIPN. While acute and chronic OXAIPN appear to have distinctpathophysiology, studies have shown that patients with more acutesymptoms eventually develop more severe chronic OXAIPN [Argyriou 2013].The clinical characteristics, mechanisms and management (includingdiagnosis, prevention and treatment) of platinum-induced peripheralneuropathy and CIPN (chemotherapy induced peripheral neuropathy) aremeticulously reviewed in [Hershman 2014, Argriou 2015, Argriou 2012,Avan 2015, Miltenburg 2014, Han 2013, Seretny 2014]. CIPN assessment,using patient reported outcome (PRO) based criteria, such as NCI-CTCv3.0, EORTC QLQ-CIPN20 and TNSc, and subjective quantitativemeasurements, such as nerve conduction studies and quantitative sensorytesting (QST), are reviewed in [Cavaletti 2013, Griffth 2014].

Various agents have been proposed to prevent and/or treat CIPN. Theseinclude antioxidants, sodium channel blockers, opioid receptoranalgesics, nerve growth factor (NGF) modulators, tricyclicantidepressants, neurotrophic agents, metal chelators, anticonvulsants(collectively reviewed in [Avan 2015]), sigma receptor ligands[US2011/0052723] and EGFR inhibitors [US 2015/0320861].

Despite CIPN being relatively distinct from other forms of neuropathicpain in many ways, including pathophysiology and symptoms [Hershman2014, Wolf 2012], many of the agents aforementioned either have a recordof efficacy for some neuropathic pain conditions or target painsignaling pathways. For example, tricyclic antidepressants amitriptylineand nortriptyline, which are also potent sigma receptor ligands[US2011/0052723, Werling 2007], are effective on various forms ofneuropathic pains, but was ineffective on preventing nor treating PNsymptoms induced by chemotherapeutic drugs including cisplatin andoxaliplatin [Kautio 2008, Kautio 2008].

In other clinical studies, antioxidant vitamin E, which was thought tobe a protector against platinum assault in DRG, failed to reduce theincidence of sensory neuropathy in patients treated with platinum drugs[Pace 2003, Kottschade 2011].

After meticulous review of most clinical studies on agents for managingCIPN, American Society of Clinical Oncology (ASCO) concluded, in itscritical 2014 guideline for CIPN prevention and management [Hershman2014], “There are no established agents recommended for the preventionof CIPN in patients with cancer undergoing treatment with neurotoxicagents.”

It has been long discovered and generally accepted that unwanted highaccumulation of toxic platinum drugs in DRG is the primary mechanism ofDRG nerve damage and hence CIPN related to treatment with platinum drugs[Miltenburg 2014, Holmes 1998]. A recent study suggests that oxaliplatinmay accumulate in DRG cells via Organic Cation Transporter 2 (OCT2,SLC22A2), which is highly expressed in DRG neurons [Sprowl 2013].Studies have shown several platinum agents, such as cisplatin,oxaliplatin and tetraplatin, are OCT2 substrates. OCT2 is also highlyexpressed in renal proximal tubule epithelium and cochlear epithelium[Hellberg 2015], potentially facilitating the uptake platinum agents andthus aggravating the cytotoxicity of cisplatin in these cells.

Besides OCT2, cisplatin and oxaliplatin are also transported by othertransporters. Some of these transporters may potentiate anti-tumorefficacy; others may protect non-tumorous tissues from platinum toxicity[Burger 2011, Harrach 2015, Tashima 2015, Li 2014, Nakanishi 2007].Therefore, it is critical to have a selective OCT2 inhibitor withdesirable pharmacokinetic and safety profiles in order for it to be usedclinically for minimizing cisplatin and oxaliplatin toxicities in normaltissues, without compromising their anti-tumor effectiveness.

SUMMARY

The present disclosure meets the need for reduced platinum drug-inducedtoxicity by providing methods for reducing platinum drug-inducedtoxicity in a cell expressing Organic Cation Transporter 2 (OCT2), forreducing platinum drug-induced toxicity in a subject, for treatingcancer in a subject, and for increasing efficacy of platinum treatmentin a subject, which include the step of providing an OCT2 inhibitor to acell expressing OCT2 or to a subject comprising cells expressing OCT2.The present disclosure also meets the need for reduced platinumdrug-induced toxicity by providing pharmaceutical compositionscomprising a platinum drug and an OCT2 inhibitor.

The teachings herein demonstrate the discovery that inhibitors of OCT2can reduce uptake of platinum drugs into healthy, non-cancer cells whileleaving cancer cells, and the therapeutic toxic effect of platinum drugson cancer cells, unaffected. FIG. 14 illustrates the inventive concept.OCT2 is critical for cellular uptake of platinum derivatives in certaincell types including kidney cells, neuronal cells, more specificallydorsal root ganglion on afferent neuronal cells, and cochlear cells.Therefore, OCT2 inhibitor provided to a non-cancerous kidney proximaltubule cell reduces the amount of platinum drug entering into the cell,and thus reduces platinum drug-induced toxicity in the cell byinhibiting uptake of platinum drug through OCT2. The contribution ofother transporters like CTR1 to the uptake of platinum derivatives bykidney proximal tubule cells has not been validated yet. A similarscenario is expected to occur in Dorsal Root Ganglions where OCT2 isthought to be responsible for platinum accumulation and neurotoxicitywith unknown contributions from other transporters. In contrast, if acolorectal cancer cell does not express OCT2, then an OCT2 inhibitor hasno effect on this cell. Instead, the colorectal cancer cell typicallyexpresses two other transporters, OCT1 and OCT3, that could mediateuptake of platinum drug into the cell, thereby allowing the platinumdrug to remain therapeutically effective with respect to the cancercell. If OCT2 is present on cancer cells and blocked by an OCT2inhibitor, it is expected that the contributions of both OCT1 and 3 willbe sufficient to preserve the uptake of platinum derivatives andtherefore their anti-tumor activity. It is not known if othertransporters are critical for the uptake of platinum derivatives intumor cells.

Thus one aspect of the present disclosure includes methods for reducingplatinum drug-induced toxicity in a cell expressing Organic CationTransporter 2 (OCT2) comprising providing a platinum drug to the cellexpressing OCT2; and providing an OCT2 inhibitor that reducesOCT2-mediated platinum drug uptake into the cell, wherein the OCT2inhibitor is buflomedil or a buflomedil salt and reduces platinum druginduced toxicity in the cell.

Another aspect includes methods for reducing platinum drug-inducedtoxicity in a cell expressing Organic Cation Transporter 2 (OCT2)comprising providing a platinum drug to the cell expressing OCT2; andproviding an OCT2 inhibitor that reduces OCT2-mediated platinum druguptake into the cell, wherein the OCT2 inhibitor is dolutegravir or adolutegravir salt and reduces platinum drug-induced toxicity in thecell.

Another aspect includes methods for reducing platinum drug-inducedtoxicity in a cell expressing Organic Cation Transporter 2 (OCT2)comprising providing a platinum drug to the cell expressing OCT2; andproviding an OCT2 inhibitor that reduces OCT2-mediated platinum druguptake into the cell, wherein the OCT2 inhibitor comprises imidazole andreduces platinum drug-induced toxicity in the cell. In some embodiments,the OCT2 inhibitor is miconazole or a salt thereof.

In some embodiments of the above aspects, the platinum drug and the OCT2inhibitor are provided at the same time. In some embodiments, the OCT2inhibitor is provided before the platinum drug. In some embodiments, theOCT2 inhibitor is provided after the platinum drug. In some embodiments,the cell expressing OCT2 is a cell selected from the group consisting ofkidney cell, neuron cell, ear cell, and blood cell. In some embodiments,the cell is a sensory neuron cell. In some embodiments, the cell is akidney cell. In some embodiments, the platinum drug is oxaliplatin. Insome embodiments, the platinum drug is cisplatin. In some embodiments,the platinum drug is tetraplatin. In some embodiments, OCT2-mediatedplatinum drug uptake into the cell expressing OCT2 is inhibited by atleast 50 percent, at least 70 percent or at least 90 percent as comparedto OCT2-mediated platinum drug uptake into a cell not in the presence ofthe OCT2 inhibitor.

Another aspect of the present disclosure includes methods for reducingplatinum drug-induced toxicity in a subject comprising providing aplatinum drug to a subject comprising cells expressing Organic CationTransporter 2 (OCT2); and providing an OCT2 inhibitor to the subject,wherein the OCT2 inhibitor is buflomedil or a buflomedil salt andreduces OCT2-mediated platinum drug uptake into the cells expressingOCT2, thereby reducing platinum drug-induced toxicity in the subject.

Another aspect includes methods for reducing platinum drug inducedtoxicity in a subject comprising providing a platinum drug to a subjectcomprising cells expressing Organic Cation Transporter 2 (OCT2); andproviding an OCT2 inhibitor to the subject, wherein the OCT2 inhibitoris dolutegravir or a dolutegravir salt and reduces OCT2 mediatedplatinum drug uptake into the cells expressing OCT2, thereby reducingplatinum drug induced toxicity in the subject.

Another aspect includes methods for reducing platinum drug-inducedtoxicity in a subject comprising providing a platinum drug to a subjectcomprising cells expressing Organic Cation Transporter 2 (OCT2); andproviding an OCT2 inhibitor to the subject, wherein the OCT2 inhibitorcomprises imidazole and reduces OCT2-mediated platinum drug uptake intothe cells expressing OCT2, thereby reducing platinum drug-inducedtoxicity in the subject. In some embodiments, the OCT2 inhibitor ismiconazole or a salt thereof.

Another aspect of the present disclosure includes methods for reducingplatinum drug-induced toxicity in a subject, comprising providing anOCT2 inhibitor to a subject comprising cells expressing OCT2; whereinthe OCT2 inhibitor is buflomedil or a buflomedil salt, the subject hasbeen or will be provided with platinum drug and the OCT2 inhibitorreduces OCT2-mediated platinum drug uptake into the cells expressingOCT2, thereby reducing platinum drug-induced toxicity in the subject.

Another aspect includes methods for reducing platinum drug inducedtoxicity in a subject, comprising providing an OCT2 inhibitor to asubject comprising cells expressing OCT2; wherein the OCT2 inhibitor isdolutegravir or a dolutegravir salt, the subject has been or will beprovided with platinum drug and the OCT2 inhibitor reduces OCT2 mediatedplatinum drug uptake into the cells expressing OCT2, thereby reducingplatinum drug induced toxicity in the subject.

Another aspect includes methods for reducing platinum drug-inducedtoxicity in a subject, comprising providing an OCT2 inhibitor to asubject comprising cells expressing OCT2; wherein the OCT2 inhibitorcomprises imidazole, the subject has been or will be provided withplatinum drug and the OCT2 inhibitor reduces OCT2-mediated platinum druguptake into the cells expressing OCT2, thereby reducing platinumdrug-induced toxicity in the subject. In some embodiments, the OCT2inhibitor is miconazole or a salt thereof.

In some embodiments of the above aspects, the platinum drug and the OCT2inhibitor are provided at the same time. In some embodiments, the OCT2inhibitor is provided before the platinum drug. In some embodiments, theOCT2 inhibitor is provided after the platinum drug. In some embodiments,the platinum drug is oxaliplatin. In some embodiments, the platinum drugis cisplatin. In some embodiments, the platinum drug is tetraplatin. Insome embodiments, the amount of OCT2 inhibitor provided to the subjectis at a less than a therapeutically effective dosage. In someembodiments, the amount of OCT2 inhibitor provided to the subject is 10mg to 2000 mg per day. In some embodiments, the amount of platinum drugprovided to the subject during one treatment session is greater thanwhat is provided under standard clinical practices. In some embodiments,the cumulative amount of platinum drug provided to the subject over theentire course of treatment is greater than what is provided understandard clinical practices. In some embodiments, the platinum drug isprovided at a greater frequency than under standard clinical practices.In some embodiments, the platinum drug-induced toxicity is selected fromthe group consisting of nephrotoxicity, neurotoxicity, hematoxicity, andototoxicity. In some embodiments, the platinum drug-induced toxicity isneurotoxicity. In some embodiments, the neurotoxicity is peripheralneuropathy. In some embodiments, the peripheral neuropathy is Grade 3 orGrade 4 peripheral neuropathy. In some embodiments, the platinumdrug-induced toxicity is nephrotoxicity. In some embodiments, thesubject is a human or a non-human animal. In some embodiments, the OCT2inhibitor is provided enterally, intravenously, intramuscularly,intraperitoneally, orally, or parenterally. In some embodiments,prevalence of platinum drug-induced toxicity in a group of subjects isreduced by at least 10% as compared to the prevalence of platinumdrug-induced toxicity in a group of subjects not provided with an OCT2inhibitor.

Another aspect of the present disclosure includes methods for treatingcancer in a subject comprising providing a therapeutically effectiveamount of platinum drug to the subject having cancer, wherein thesubject comprises a cancerous cell expressing platinum drug uptaketransporters not inhibited by an Organic Cation Transporter 2 (OCT2)inhibitor; and providing an OCT2 inhibitor to the subject, wherein theOCT2 inhibitor is buflomedil or a buflomedil salt, thereby treatingcancer in the subject.

Another aspect includes methods for treating cancer in a subjectcomprising providing a therapeutically effective amount of platinum drugto the subject having cancer, wherein the subject comprises a cancerouscell expressing platinum drug uptake transporters not inhibited by anOrganic Cation Transporter 2 (OCT2) inhibitor; and providing an OCT2inhibitor to the subject, wherein the OCT2 inhibitor is dolutegravir ora dolutegravir salt, thereby treating cancer in the subject.

Another aspect includes methods for treating cancer in a subjectcomprising providing a therapeutically effective amount of platinum drugto the subject having cancer, wherein the subject comprises a cancerouscell expressing platinum drug uptake transporters not inhibited by anOrganic Cation Transporter 2 (OCT2) inhibitor; and providing an OCT2inhibitor to the subject, wherein the OCT2 inhibitor comprisesimidazole, thereby treating cancer in the subject. In some embodiments,the OCT2 inhibitor is miconazole or a salt thereof.

Another aspect includes methods for increasing efficacy of platinum drugtreatment in a subject comprising providing a therapeutically effectiveamount of platinum drug to the subject having cancer, wherein thesubject comprises a cancerous cell expressing platinum drug uptaketransporters not inhibited by an OCT2 inhibitor; and providing an OCT2inhibitor to the subject, wherein the OCT2 inhibitor is buflomedil or abuflomedil salt, thereby increasing efficacy of the platinum drugtreatment.

Another aspect includes methods for increasing efficacy of platinum drugtreatment in a subject comprising providing a therapeutically effectiveamount of platinum drug to the subject having cancer, wherein thesubject comprises a cancerous cell expressing platinum drug uptaketransporters not inhibited by an OCT2 inhibitor; and providing an OCT2inhibitor to the subject, wherein the OCT2 inhibitor is dolutegravir ora dolutegravir salt, thereby increasing efficacy of the platinum drugtreatment.

Another aspect includes methods for increasing efficacy of platinum drugtreatment in a subject comprising providing a therapeutically effectiveamount of platinum drug to the subject having cancer, wherein thesubject comprises a cancerous cell expressing platinum drug uptaketransporters not inhibited by an OCT2 inhibitor; and providing an OCT2inhibitor to the subject, wherein the OCT2 inhibitor comprisesimidazole, thereby increasing efficacy of the platinum drug treatment.In some embodiments, the OCT2 inhibitor is miconazole or a salt thereof.

In some embodiments of the above aspects, the platinum drug and the OCT2inhibitor are provided at the same time. In some embodiments, the OCT2inhibitor is provided before the platinum drug. In some embodiments, theOCT2 inhibitor is provided after the platinum drug. In some embodiments,the platinum drug is oxaliplatin. In some embodiments, the platinum drugis cisplatin. In some embodiments, the platinum drug is tetraplatin. Insome embodiments, the amount of OCT2 inhibitor provided to the subjectis at a less than a therapeutically effective dosage. In someembodiments, the amount of OCT2 inhibitor provided to the subject is 10mg to 2000 mg per day. In some embodiments, the amount of platinum drugprovided to the subject during one treatment session is greater thanwhat is provided under standard clinical practices. In some embodiments,the cumulative amount of platinum drug provided to the subject over theentire course of treatment is greater than what is provided understandard clinical practices. In some embodiments, the platinum drug isprovided at a greater frequency than under standard clinical practices.

In some embodiments, the cancer is selected from the group consisting ofadenocarcinoma of the pancreas, ampullary and periampullary carcinoma,adenocarcinoma of the anus, appendiceal carcinoma, hepatocellularcarcinoma, carcinoma of the colon or rectum, epithelial ovariancarcinoma, fallopian tube carcinoma. primary peritoneal cancer,esophageal or esophagogastric junction carcinoma, gastric carcinoma,small bowel carcinoma, testicular cancer, cholangiocarcinoma, pancreaticadenocarcinoma, carcinoma of unknown primary origin, chronic lymphocyticleukemia/small lymphocytic lymphoma, non-Hodgkin's lymphoma, adultT-cell leukemia/lymphoma, AIDS-related B-cell lymphoma, diffuse largeB-cell lymphoma, follicular lymphoma, gastric MALT lymphoma, nongastricMALT lymphoma, mantle cell lymphoma, mycosis fungoides/Sezary syndrome,splenic marginal zone lymphoma, peripheral T cell lymphoma, primarycutaneous B-cell lymphoma, primary cutaneous anaplastic large celllymphoma (ALCL), lung cancer, liver cancer, and gallbladder cancer. Insome embodiments, the cancer is carcinoma of the colon or rectum. Insome embodiments, the cancer is liver cancer. In some embodiments, thecancer is lung cancer.

In some embodiments, the platinum drug uptake transporters not inhibitedby an OCT2 inhibitor are Organic Cation Transporter 1 (OCT1) uptaketransporters. In some embodiments, the platinum drug uptake transportersnot inhibited by an OCT2 inhibitor are Organic Cation Transporter 3(OCT3) uptake transporters. In some embodiments, the platinum druguptake transporters not inhibited by an OCT2 inhibitor are CopperTransporter I (CTR1) uptake transporters.

In some embodiments, the method further comprises the step ofdetermining whether the cancerous cell expresses platinum drug uptaketransporters not inhibited by an OCT2 inhibitor. In some embodiments,the method further comprises providing a therapeutically effectiveamount of one or more additional chemotherapeutic agents to the subjectin addition to the platinum drug. In some embodiments, the one or moreadditional chemotherapeutic agents is selected from the group consistingof 5-fluorouracil, bevacizumab, capecetabine, gemcitabine, irinotecan,and leucovorin. In some embodiments, the subject is a human or anon-human animal. In some embodiments, the OCT2 inhibitor is providedenterally, intravenously, intramuscularly, intraperitoneally, orally, orparenterally.

In another aspect the present disclosure includes pharmaceuticalcompositions comprising a platinum drug, an OCT2 inhibitor, wherein theOCT2 inhibitor is buflomedil or a buflomedil salt; and apharmaceutically acceptable carrier. Another aspect includespharmaceutical compositions comprising a platinum drug, an OCT2inhibitor, wherein the OCT2 inhibitor is dolutegravir or a dolutegravirsalt; and a pharmaceutically acceptable carrier. Another aspect includespharmaceutical compositions comprising a platinum drug, an OCT2inhibitor, wherein the OCT2 inhibitor comprises imidazole; and apharmaceutically acceptable carrier. In some embodiments, the OCT2inhibitor is miconazole or a salt thereof.

In some embodiments of the above aspects, the amount of platinum drug isgreater than what is present in a standard pharmaceutical compositioncomprising platinum drug. In some embodiments, the pharmaceuticalcompositions further comprise a therapeutically effective amount of oneor more additional chemotherapeutic agents in addition to the platinumdrug. In some embodiments, the one or more additional chemotherapeuticagents is selected from the group consisting of 5-fluorouracil,bevacizumab, capecetabine, gemcitabine, irinotecan, and leucovorin. Insome embodiments, the platinum drug is oxaliplatin. In some embodiments,the platinum drug is cisplatin. In some embodiments, the platinum drugis tetraplatin.

In some embodiments of the aspects where the OCT2 inhibitor comprisesimidazole, the OCT2 inhibitor has a C_(max,u)/IC₅₀ orC_(max)/IC_(50,app) of at least 1. In some embodiments, the OCT2inhibitor has a C_(max,u)/IC₅₀ or C_(max)/IC_(50,app) of at least 3. Insome embodiments, the OCT2 inhibitor is not toxic to the cell at itsclinical concentration. In some embodiments, the OCT2 inhibitor does notreduce anti-cancer activity of the platinum drug at its clinicalconcentration by more than 20% as compared to anti-cancer activity ofthe platinum drug at its clinical concentration in the absence of theOCT2 inhibitor. In some embodiments, the OCT2 inhibitor does not reduceuptake of the platinum drug at its clinical concentration into the cellvia other transporters by more than 20% as compared to uptake of theplatinum drug at its clinical concentration via other transporters inthe absence of the OCT2 inhibitor. In some embodiments, the OCT2inhibitor does not reduce the efflux of the platinum drug at itsclinical concentration by more than 20% as compared to the efflux of theplatinum drug at its clinical concentration in the absence of the OCT2inhibitor. In some embodiments, the OCT2 inhibitor has a mean half-lifethat is greater than 2 hours.

Another aspect of the present disclosure includes methods of predictingthe efficacy of a platinum drug and Organic Cation Transporter 2 (OCT2)inhibitor therapy in a subject having cancer comprising obtaining asample comprising at least one cancerous cell; and determining whetherthe cancerous cell expresses platinum drug uptake transporters notinhibited by an OCT2 inhibitor, wherein platinum drug and OCT2 inhibitortherapy is likely to be effective in a subject where the cancerous cellexpresses platinum drug uptake transporters not inhibited by an OCT2inhibitor.

Another aspect includes methods of predicting the efficacy of platinumdrug and Organic Cation Transporter 2 (OCT2) inhibitor therapy in asubject having cancer comprising obtaining a sample comprising at leastone cancerous cell; and determining whether the cancerous cell expressesOCT2, wherein the platinum drug and OCT2 inhibitor therapy is not likelyto be effective in a subject where the cancerous cell primarilyexpresses OCT2.

In some embodiments of the above aspects, the platinum drug isoxaliplatin. In some embodiments, the platinum drug is cisplatin. Insome embodiments, the platinum drug is tetraplatin. In some embodiments,the OCT2 inhibitor therapy comprises administering buflomedil or abuflomedil salt. In some embodiments, the OCT2 inhibitor therapycomprises administering dolutegravir or a dolutegravir salt. In someembodiments, the OCT2 inhibitor therapy comprises administering an OCT2inhibitor comprising imidazole. In some embodiments, the OCT2 inhibitortherapy comprises administering an OCT2 inhibitor comprising miconazoleor a salt thereof.

The present disclosure provides a method for reducing platinumdrug-induced neurotoxicity in a subject in need thereof comprisingadministering a platinum drug that is oxaliplatin to the subject in needthereof; and administering an effective dose of a selective OrganicCation Transporter 2 (OCT2) inhibitor to the subject in need thereof,wherein the subject in need thereof has a cancer.

The present disclosure provides a method for treating cancer in asubject in need thereof comprising administering a therapeuticallyeffective amount of a platinum drug that is oxaliplatin to the subjectin need thereof, and administering an effective dose of a selectiveOrganic Cation Transporter 2 (OCT2) inhibitor to the subject in needthereof.

The present disclosure provides a method for increasing patientcompliance for treating cancer in a subject in need thereof comprisingadministering a therapeutically effective amount of a platinum drug thatis oxaliplatin to the subject in need thereof; and administering aselective Organic Cation Transporter 2 (OCT2) inhibitor to the subjectin need thereof in a dose effective to reduce platinum drug-inducedneurotoxicity in said subject in need thereof, whereby said subjectcompletes treatment with a cumulative dose at least 500 mg/m².

In some embodiments, the therapeutically effective amount of platinumdrug administered is known to cause platinum drug-induced neurotoxicityin subjects. In some embodiments, the neurotoxicity is peripheralneurotoxicity. In some embodiments, the neurotoxicity is damage to asensory neuron. In some embodiments, the neurotoxicity is damage to amotor neuron. In some embodiments, the neurotoxicity is chronicneurotoxicity. In some embodiments, the neurotoxicity is acute syndrometransient neurotoxicity. In some embodiments, the neurotoxicity occurs 1hour to seven days after first treatment. In some embodiments, theneurotoxicity occurs after a subject completes treatment with acumulative dose of at least 500 mg/m². In some embodiments, theneurotoxicity is damage to dorsal root ganglia (DRG). In someembodiments, the dose of selective OCT2 inhibitor is effective tominimize platinum drug-induced neurotoxicity in said subject in needthereof.

In some embodiments, the dose of selective OCT2 inhibitor is effectiveto prevent platinum drug-induced neurotoxicity of Grade 3 or higher, asassessed by a method selected from National Cancer Institute-CommonToxicity Criteria (NCI-CTC) sensory scale, National CancerInstitute-Common Toxicity Criteria (NCI-CTC) motor scale, TotalNeuropathy Score clinical version (TNSc) and European Organization forResearch and Treatment of Cancer CIPN specific self-report questionnaire(EORTC QOL-CIPN20), in patients treated with a cumulative dose of atleast 100 mg/m². In some embodiments, the dose of selective OCT2inhibitor is effective to prevent platinum drug-induced neurotoxicity ofGrade 3 or higher, as assessed by a method selected from National CancerInstitute-Common Toxicity Criteria (NCI-CTC) sensory scale, NationalCancer Institute-Common Toxicity Criteria (NCI-CTC) motor scale, TotalNeuropathy Score clinical version (TNSc) and European Organization forResearch and Treatment of Cancer CIPN specific self-report questionnaire(EORTC QOL-CIPN20), in patients treated with a platinum drug doseintensity of at least 30 mg/week/m². In some embodiments, the dose ofselective OCT2 inhibitor is effective to prevent platinum drug-inducedneurotoxicity of Grade 3 or higher, as assessed by a method selectedfrom National Cancer Institute-Common Toxicity Criteria (NCI-CTC)sensory scale, National Cancer Institute-Common Toxicity Criteria(NCI-CTC) motor scale, Total Neuropathy Score clinical version (TNSc)and European Organization for Research and Treatment of Cancer CIPNspecific self-report questionnaire (EORTC QOL-CIPN20), in patientstreated with a platinum drug dose of at least 80 mg/m².

In some embodiments, the method further comprises assessing platinumdrug-induced neurotoxicity in a subject after administration of theplatinum drug. In some embodiments, the neurotoxicity is assessed by amethod selected from National Cancer Institute-Common Toxicity Criteria(NCI-CTC) sensory scale, National Cancer Institute-Common ToxicityCriteria (NCI-CTC) motor scale, Total Neuropathy Score clinical version(TNSc) and European Organization for Research and Treatment of CancerCIPN specific self-report questionnaire (EORTC QOL-CIPN20). In someembodiments, the neurotoxicity is assessed by a measurement selectedfrom sensory nerve action potential, sensory nerve conduction velocity,cold pain threshold, heat pain threshold, mechanical pain threshold,cold detection threshold, warm detection threshold, mechanical detectionthreshold, vibration perception threshold, current perception threshold,pinprick sensibility, deep tendon reflexes and grip strength. In someembodiments, the neurotoxicity is assessed by measuring sensory nerveaction potential in one of radial, dorsal sural, sural and ulnar nerves.In some embodiments, the method further comprises establishing thesubject's baseline prior to administration of the platinum drug. In someembodiments, the platinum drug-induced neurotoxicity is assessed nearthe midpoint of treatment. In some embodiments, the platinumdrug-induced neurotoxicity is assessed after treatment with a cumulativedose of 200 mg/m².

In some embodiments, the selective OCT2 inhibitor is selected from thegroup consisting of buflomedil, a buflomedil salt, dolutegravir, and adolutegravir salt. In some embodiments, the selective OCT2 inhibitor isbuflomedil, or a buflomedil salt. In some embodiments, the dose ofbuflomedil or a buflomedil salt administered in a subject is adjustedbased on at least one of the factors of the said subject, body weight,body surface area, height, age, gender, alcohol use, tobacco use, lifestyle, renal function, liver function, genetic polymorphism andco-medications. In some embodiments, the dose of buflomedil or abuflomedil salt results in its plasma level during the period ofplatinum administration at least 0.43 mg/l, 0.86 mg/l, 1.29 mg/l, 1.72mg/l or 2.15 mg/l. In some embodiments, the dose of buflomedil or abuflomedil salt is at least 100 mg, 200 mg, 300 mg, 400 mg, 500 mg or600 mg. In some embodiments, the dose of buflomedil or a buflomedil saltis at least 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 8mg/kg or 10 mg/kg.

In some embodiments, the selective OCT2 inhibitor is dolutegravir or adolutegravir salt. In some embodiments, the dose of dolutegravir or adolutegravir salt results in its plasma level during the period ofplatinum administration at least 1.4 mg/l, 2.8 mg/l, 4.2 mg/l, 5.6 mg/lor 7.0 mg/l. In some embodiments, the dose of dolutegravir or adolutegravir salt is at least 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300mg or 400 mg. In some embodiments, the dose of dolutegravir or adolutegravir salt is at least 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5mg/kg or 6 mg/kg.

In some embodiments, the selective OCT2 inhibitor does not reduce theefficacy of the platinum drug. In some embodiments, the selective OCT2inhibitor is administered at a dose that results in its plasmaconcentration during the period of platinum drug administration lessthan its maximum tolerated plasma concentration (MTC) and greater than1×, 2×, 3×, 4× of its IC₅₀ value for OCT2-mediated transport of 20 μMoxaliplatin assessed in human serum or an assay buffer containing 4%bovine serum albumin. In some embodiments, the selective OCT2 inhibitorhas an ICs for OCT2-mediated transport of 20 μM oxaliplatin in humanserum or an assay solution containing 4% bovine serum albumin, of lessthan or equal to 2 μM. In some embodiments, the selective OCT2 inhibitorhas an IC₅₀ for OCT2-mediated transport of 20 μM oxaliplatin in humanserum or an assay solution containing 4% bovine serum albumin that is atleast 15-fold less than the selective OCT2 inhibitor IC₅₀ for 20 μMoxaliplatin transport in human serum or an assay solution containing 4%bovine serum albumin mediated by OCT1-, OCT-3, and MATE-1.

In some embodiments, the subject in need thereof has a cancer expressingat least one of Organic Cation Transporter 1 (OCT1) or Organic CationTransporter 3 (OCT3). In some embodiments, the selective OCT2 inhibitoris buflomedil, or a buflomedil salt. In some embodiments, the selectiveOCT2 inhibitor is dolutegravir, or a dolutegravir salt. In someembodiments, the platinum drug and the selective OCT2 inhibitor areadministered at the same time. In some embodiments, the selective OCT2inhibitor is administered before the platinum drug. In some embodiments,the selective OCT2 inhibitor is administered after the platinum drug. Insome embodiments, the amount of platinum drug administered to thesubject during one treatment session is greater than what isadministered under standard clinical practices. In some embodiments, thecumulative amount of platinum drug administered to the subject in needthereof over the entire course of treatment is greater than what isadministered under standard clinical practices. In some embodiments, theplatinum drug is administered at a greater frequency than under standardclinical practices.

In some embodiments, the neurotoxicity is peripheral neuropathy. In someembodiments, the peripheral neuropathy is Grade 3 or Grade 4 peripheralneuropathy. In some embodiments, the subject in need thereof is a humanor a non-human animal.

In some embodiments, the selective OCT2 inhibitor is administeredenterally, intravenously, intramuscularly, intraperitoneally, orally, orparenterally. In some embodiments, the selective OCT2 inhibitor isadministered via more than one route of administration. In someembodiments, the platinum drug and the selective OCT2 inhibitor areadministered via the same route of administration. In some embodiments,the selective OCT2 inhibitor is administered via intravenous infusion.In some embodiments, the selective OCT2 inhibitor is administrated viaintravenous injection and intravenous infusion. In some embodiments,intravenous infusion is over a period of time at least 1 hour. In someembodiments, the rate of intravenous infusion is constant. In someembodiments, the rate of intravenous infusion is variable.

In some embodiments, the cancer expresses OCT1. In some embodiments, thecancer expresses OCT3. In some embodiments, the cancer expresses OCT1and OCT3. In some embodiments, the cancer is selected from the groupconsisting of adenocarcinoma of the pancreas, ampullary andperiampullary carcinoma, adenocarcinoma of the anus, appendicealcarcinoma, hepatocellular carcinoma, carcinoma of the colon or rectum,epithelial ovarian carcinoma, fallopian tube carcinoma. primaryperitoneal cancer, esophageal or esophagogastric junction carcinoma,gastric carcinoma, small bowel carcinoma, testicular cancer,cholangiocarcinoma, pancreatic adenocarcinoma, carcinoma of unknownprimary origin, chronic lymphocytic leukemia/small lymphocytic lymphoma,non-Hodgkin's lymphoma, adult T-cell leukemia/lymphoma, AIDS-relatedB-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma,gastric MALT lymphoma, nongastric MALT lymphoma, mantle cell lymphoma,mycosis fungoides/Sezary syndrome, splenic marginal zone lymphoma,peripheral T cell lymphoma, primary cutaneous B-cell lymphoma, primarycutaneous anaplastic large cell lymphoma (ALCL), lung cancer, livercancer, head and neck cancer, prostate cancer, smooth muscle cancer andgallbladder cancer. In some embodiments, the cancer is ovarian cancer.In some embodiments, the cancer is head and neck cancer. In someembodiments, the cancer is prostate cancer. In some embodiments, thecancer is lymphoma. In some embodiments, the cancer is smooth musclecancer. In some embodiments, the cancer is carcinoma of the colon orrectum. In some embodiments, the cancer is liver cancer. In someembodiments, the cancer is lung cancer.

In some embodiments, the method further comprises administering to thesubject in need thereof a therapeutically effective amount of one ormore additional cancer chemotherapeutic agents in addition to theplatinum drug. In some embodiments, the one or more additionalchemotherapeutic agents is selected from the group consisting of5-fluorouracil, bevacizumab, capecetabine, gemcitabine, irinotecan, andleucovorin.

The present disclosure provides a pharmaceutical composition formulatedfor intravenous administration comprising a platinum drug that isoxaliplatin, a selective OCT2 inhibitor selected from the groupconsisting of buflomedil, a buflomedil salt, dolutegravir, and adolutegravir salt, and a pharmaceutically acceptable carrier.

In some embodiments, the selective OCT2 inhibitor is buflomedil, or abuflomedil salt. In some embodiments, the OCT2 inhibitor isdolutegravir, or a dolutegravir salt. In some embodiments, the amount ofthe platinum drug is greater than what is present in a standardpharmaceutical composition comprising the platinum drug. In someembodiments, the pharmaceutical composition further comprises atherapeutically effective amount of one or more additional cancerchemotherapeutic agents in addition to the platinum drug. In someembodiments, the one or more additional chemotherapeutic agents isselected from the group consisting of 5-fluorouracil, bevacizumab,capecetabine, gemcitabine, irinotecan, and leucovorin.

The present disclosure provides a kit comprising a therapeuticallyeffective amount of a platinum drug that is oxaliplatin; a selectiveOCT2 inhibitor and instructions for use. In some embodiments, the kitfurther comprises instructions for determining a desirable dose of theselective OCT2 inhibitor for a subject in need. In some embodiments, thedose is determined based on at least one factor of the said subjectselected from body weight, body surface area, height, age, gender,alcohol use, tobacco use, life style, renal function, liver function,genetic polymorphism and co-medications. In some embodiments, the doseis determined by monitoring plasma level of the OCT2 inhibitor in thesaid subject. In some embodiments, the selective OCT2 inhibitor isbuflomedil or a buflomedil salt. In some embodiments, buflomedil or abuflomedil salt is present in an amount greater than 300 mg, 450 mg, 600mg, 800 mg or 1000 mg. In some embodiments, the selective OCT2 inhibitoris dolutegravir or a dolutegravir salt. In some embodiments,dolutegravir or a dolutegravir salt is present in an amount greater than50 mg, 75 mg, 100 mg, 150 mg, 200 mg or 300 mg. In some embodiments, theinstructions state that the kit is intended for use in reducing platinumdrug-induced neurotoxicity. In some embodiments, the instructions statethat the kit is intended for use in treating cancer. In someembodiments, the kit further comprises a therapeutically effectiveamount of one or more additional cancer chemotherapeutic agents inaddition to the platinum drug. In some embodiments, the one or moreadditional chemotherapeutic agents is selected from the group consistingof 5-fluorouracil, bevacizumab, capecetabine, gemcitabine, irinotecan,and leucovorin.

The present disclosure provides a method for reducing platinumdrug-induced toxicity in a subject in need thereof comprisingadministering a platinum drug that is cisplatin to the subject in needthereof; and administering an effective dose of a selective OrganicCation Transporter 2 (OCT2) inhibitor to the subject in need thereof,wherein the toxicity is nephrotoxicity, ototoxicity or peripheralneuropathy, and wherein the subject in need thereof has a cancer.

The present disclosure provides a method for treating cancer in asubject in need thereof comprising administering a therapeuticallyeffective amount of a platinum drug that is cisplatin to the subject inneed thereof; and administering an effective dose of a selective OrganicCation Transporter 2 (OCT2) inhibitor to the subject in need thereof.

The present disclosure provides a method for increasing patientcompliance for treating cancer in a subject in need thereof comprisingadministering a therapeutically effective amount of a platinum drug thatis cisplatin to the subject in need thereof; and administering aselective Organic Cation Transporter 2 (OCT2) inhibitor to the subjectin need thereof in a dose effective to reduce platinum drug-inducedtoxicity in said subject in need thereof, whereby said subject completestreatment with a cumulative dose of at least 100 mg/m².

The present disclosure provides a method for reducing platinumdrug-induced toxicity in a subject in need thereof comprisingdetermining an effective dose to achieve a plasma level of a selectiveOrganic Cation Transporter 2 (OCT2) inhibitor that is buflomedil or abuflomedil salt in the subject in need thereof, wherein the plasma levelis two to five times IC₅₀ of the selective Organic Cation Transporter 2(OCT2) inhibitor that is buflomedil or a buflomedil salt; administeringa platinum drug selected from the group consisting of oxaliplatin andcisplatin to the subject in need thereof; and administering an effectivedose of a selective Organic Cation Transporter 2 (OCT2) inhibitor thatis buflomedil or a buflomedil salt to the subject in need thereof,wherein the toxicity is nephrotoxicity, ototoxicity or peripheralneuropathy, and wherein the subject in need thereof has a cancer.

The present disclosure provides a method for treating cancer in asubject in need thereof comprising determining an effective dose toachieve a plasma level of a selective Organic Cation Transporter 2(OCT2) inhibitor that is buflomedil or a buflomedil salt in the subjectin need thereof, wherein the plasma level is two to five times IC₅₀ ofthe selective Organic Cation Transporter 2 (OCT2) inhibitor that isbuflomedil or a buflomedil salt; administering a therapeuticallyeffective amount of a platinum drug selected from the group consistingof oxaliplatin and cisplatin to the subject in need thereof; andadministering an effective dose of a selective Organic CationTransporter 2 (OCT2) inhibitor that is buflomedil or a buflomedil saltto the subject in need thereof.

The present disclosure provides a method for increasing patientcompliance for treating cancer in a subject in need thereof comprisingdetermining an effective dose to achieve a plasma level of a selectiveOrganic Cation Transporter 2 (OCT2) inhibitor that is buflomedil or abuflomedil salt in the subject in need thereof, wherein the plasma levelis two to five times IC₅₀ of the selective Organic Cation Transporter 2(OCT2) inhibitor that is buflomedil or a buflomedil salt; administeringa therapeutically effective amount of a platinum drug selected from thegroup consisting of oxaliplatin and cisplatin to the subject in needthereof; and administering a selective Organic Cation Transporter 2(OCT2) inhibitor that is buflomedil or a buflomedil salt to the subjectin need thereof in a dose effective to reduce platinum drug-inducedtoxicity in said subject in need thereof, whereby said subject completestreatment with a cumulative dose of at least 100 mg/m².

In some embodiments, the step of determining an effective dose toachieve a plasma level of a selective Organic Cation Transporter 2(OCT2) inhibitor is performed by administering a pre-dose of theselective Organic Cation Transporter 2 (OCT2) inhibitor. In someembodiments, the step of determining an effective dose to achieve aplasma level of a selective Organic Cation Transporter 2 (OCT2)inhibitor is determined by a reference data chart based on subjectcharacteristics.

The present disclosure provides a selective OCT2 inhibitor for use inthe treatment of cancer in a subject in need thereof, wherein the OCT2inhibitor is for use in combination with a platinum drug that isoxaliplatin. In some embodiments, the OCT2 inhibitor is foradministration at a dose capable of reducing platinum drug-inducedtoxicity.

The present disclosure provides a selective OCT2 inhibitor for use inthe treatment of cancer in a subject in need thereof, wherein the OCT2inhibitor is for use in combination with a platinum drug that iscisplatin. In some embodiments, the OCT2 inhibitor is for administrationat a dose capable of reducing platinum drug-induced toxicity.

The present disclosure provides a selective OCT2 inhibitor that isbuflomedil or a buflomedil salt for use in the treatment of cancer in asubject in need thereof, wherein the OCT2 inhibitor is for use incombination with a platinum drug that is oxaliplatin or cisplatin. Insome embodiments, the OCT2 inhibitor is for administration at a dosecapable of reducing platinum drug-induced toxicity.

The present disclosure provides a use of a selective OCT2 inhibitor inthe preparation of a medicament for the treatment of cancer, wherein themedicament is for use in combination with a platinum drug that isoxaliplatin. In some embodiments, the OCT2 inhibitor is foradministration at a dose capable of reducing platinum drug-inducedtoxicity.

The present disclosure provides a use of a selective OCT2 inhibitor inthe preparation of a medicament for the treatment of cancer, wherein themedicament is for use in combination with a platinum drug that iscisplatin. In some embodiments, the OCT2 inhibitor is for administrationat a dose capable of reducing platinum drug-induced toxicity.

The present disclosure provides a use of a selective OCT2 inhibitor thatis buflomedil or a buflomedil salt in the preparation of a medicamentfor the treatment of cancer, wherein the medicament is for use incombination with a platinum drug that is oxaliplatin or cisplatin. Insome embodiments, the OCT2 inhibitor is for administration at a dosecapable of reducing platinum drug-induced toxicity.

DESCRIPTION OF THE FIGURES

FIGS. 1 A-E depict the clinical plasma exposure of (FIG. 1A) Oxaliplatinfollowing 2 hour intravenous infusion [6]; (FIG. 1B) Miconazolefollowing intravenous injection [7]; (FIG. 1C) Buflomedil following oraladministration of 300 mg tablet q12h(1) or 600 mg slow-release tabletqd(2) [9]; (FIG. 1D) Erlotinib following oral administration of 150 mgqd [10]; and (FIG. 1E) Dolutegravir following oral administration [15].

FIG. 2 depicts the transport of oxaliplatin by different transportersexpressed in MDCK cells. (Left: oxaliplatin 20 μM in protein-free HBSS;Right: oxaliplatin 20 μM in 100% human serum).

FIGS. 3A-3D depict the inhibition of buflomedil (FIG. 3A), erlotinib(FIG. 3B), miconazole (FIG. 3C), and dolutegravir (FIG. 3D) onoxaliplatin transport in human serum or alike assay buffer by differenttransporters.

FIGS. 4A & 4B depict the inhibition of intracellular content ofoxaliplatin in cells co-expressed with OCT2, or OCT2 and MATE1, bybuflomedil (FIG. 4A) and erlotinib (FIG. 4B).

FIG. 5 depicts that buflomedil reduced cytotoxicity of 100 μMOxaliplatin in OCT2-expressing cells in a dose-dependent fashion.

FIGS. 6A-6D depict that buflomedil at 3 μm and 6 μM (FIG. 6A, FIG. 6B),and dolutegravir (DTG) at 10 μM (FIG. 6C, FIG. 6D), but not 6 uMerlotinib (FIG. 6B) and 6 μM cimetidine (FIG. 6B), significantly reducedoxaliplatin (OXA) induced cytotoxicity in OCT2 expressing cells.

FIG. 7 depicts that buflomedil significantly reduced cytotoxicity from 1mM of cisplatin in MDCK cells expressing OCT2, MATE1 and MATE2K (*indicates p<0.05).

FIGS. 8A & 8B depict that erlotinib at 6 uM (FIG. 8A) drasticallyreduced (˜8×) oxaliplatin anti-tumor potency in HT-29 cells, whereasbuflomedil at 3 μM and 6 μM (FIG. 8A), dolutegravir at 3 uM, 6 uM and 9uM (FIG. 8B) didn't affect oxaliplatin efficacy.

FIG. 9 depicts erlotinib at 3 μM significantly reduced anti-tumorpotency of oxaliplatin (p<0.05) in HT-29 cells. Whereas 6 μM buflomediland 6 μM cimetidine did not affect oxaliplatin anti-tumor activity.

FIG. 10 depicts that erlotinib, not buflomedil nor cimetidine,drastically reduced (˜10×) anti-tumor potency of oxaliplatin in HepG2cells.

FIG. 11 depicts the anti-tumor effect of cisplatin in HT-29 cells wasnot affected by co-treatment with buflomedil, erlotinib, or cimetidine.

FIGS. 12A & 12B depict that buflomedil (BFMD) at 6 uM (FIG. 12A) anddolutegravir (DTG) at 3 uM, 6 μM and 9 μM (FIG. 12B) did not affectanti-tumor potency of 5-FU in HT-29 cells.

FIGS. 13A & 13B depict that buflomedil at 6 μM did not affect anti-tumorpotency of gemcitabine in HT-29 cells.

FIG. 14 depicts inhibition of the uptake of platinum drug by selectiveOCT2 inhibitor into a kidney proximal tubule cell or a dorsal rootganglion cell, and minimal inhibition of the uptake of platinum druginto a cancer cell which primarily relies on mechanisms/transportersother than OCT2 to take up platinum drug. The contribution of severaltransporters like CTR1 to the uptake of platinum derivatives in variouscells has not been validated. The presence of OCT2 on cancer cells willvary according to the nature of tumor but the presence of OCT1 and/orOCT3 will guarantee uptake of platinum drug into the cells.

FIGS. 15A & 15B depict buflomedil exhibited no selectivity between OCT2and MATE1 when metformin is the substrate (FIG. 15A), whereas buflomedilexhibited great selectivity toward OCT2 when either cisplatin oroxaliplatin was used as the substrate, and furthermore, there wasno/minimal difference in its IC₅₀s between using oxaliplatin orcisplatin as the substrate (FIG. 15B).

FIG. 16 depicts that efflux transporters MATE1 and MATE2K was effectiveon reducing severity and potency of oxaliplatin cytotoxicity in MDCKcells.

FIGS. 17A & 17B depict plasma levels of buflomedil (FIG. 17A) anddolutegravir (FIG. 17B) in balb/c mice under different dose and route ofadministration.

FIG. 18 depicts one study design of evaluating buflomedil's effects onperipheral neuropathy in an established mouse OXAIPN model.

FIG. 19 depicts buflomedil was effective on preventing dorsal rootganglion (DRG) nerve cell injury in mice treated with 8 cycles ofoxaliplatin at 3.5 mg/kg, based on morphometric assessment of DRGsamples.

FIG. 20 depicts morphology of DRG nerve cells from histopathologysamples of mice after 8 cycles of treatment with vehicle control (topleft), oxaliplatin (bottom left), buflomedil (top right) andoxaliplatin+buflomedil (bottom right).

FIG. 21 depicts buflomedil was effective on reducing sciatic nervedamage in mice treated with 8 cycles of oxaliplatin at 3.5 mg/kg, basedon morphometric assessment of sciatic nerve samples.

FIG. 22 depicts buflomedil was effective on preventing mechanicalallodynia in mice after 8 cycles of oxaliplatin treatment.

FIG. 23 depicts buflomedil was effective on reducing cold allodynia inmice after 3 and 8 cycles of oxaliplatin treatment.

FIG. 24 depicts buflomedil was effective on reducing peripheralneurotoxicity in mice after 8 cycles of oxaliplatin treatment, based onelectrophysiological assessment of caudal nerve action potential.

FIG. 25 depicts buflomedil is effective on reducing peripheralneuropathy in mice after 8 cycles of oxaliplatin treatment, based onelectrophysiological assessment of caudal nerve conduction velocity(NCV).

FIGS. 26A & 26B depict plasma levels of buflomedil and oxaliplatin inmice 15 minutes after the 8^(th) cycle of oxaliplatin (3.5 mg/kg)treatment.

FIG. 27 depicts the design of study on the effect of dolutegravir andchlophenesin carbamate on reducing peripheral neurotoxicity after asingle oxaliplatin administration.

FIGS. 28A & 28B depict dolutegravir (FIG. 28A), but not chlophenesin(FIG. 28B), was effective on reducing mechanical hypersensitivity inmice after a single oxaliplatin treatment, based on measuring mechanicalpain threshold.

FIG. 29 depicts dolutegravir was effective on reducing coldhypersensitivity in mice after a single oxaliplatin treatment.

FIGS. 30A & 30B depict buflomedil (80 mg/kg po (orally)) was effectiveon reducing mechanical (FIG. 30A) and cold (FIG. 30B) allodynia in mice(n=3/group) after 8 cycles of oxaliplatin treatment.

FIG. 31 depicts buflomedil (80 mg/kg po) was effective on reducing nerveinjury, assessed by caudal NCV, in mice (n=3/group) after 8 cycles ofoxaliplatin treatment.

FIGS. 32A-32C depict buflomedil was effective on reducing platinumaccumulation in DRG (FIG. 32A) and kidney (FIG. 32C), but not in sciaticnerves (FIG. 32B).

FIG. 33 depicts a desirable plasma level profile of a selective OCT2inhibitor for reducing toxicity of a platinum drug while minimizing itsown adverse effect.

FIG. 34 depicts simulated buflomedil plasma level in two different groupof subjects with different reported drug clearance rate, under the samebuflomedil treatment (intravenous infusion at a constant rate of 150mg/hour, 3 hours).

DETAILED DESCRIPTION

The following description is presented to enable a person of ordinaryskill in the art to make and use the various embodiments. Descriptionsof specific devices, techniques, and applications are provided only asexamples. Various modifications to the examples described herein will bereadily apparent to those of ordinary skill in the art, and the generalprinciples defined herein may be applied to other examples andapplications without departing from the spirit and scope of the variousembodiments. Thus, the various embodiments are not intended to belimited to the examples described herein and shown, but are to beaccorded the scope consistent with the claims.

All references cited herein, including patent applications andpublications, are hereby incorporated by reference in their entirety.

Methods of Reducing Platinum Drug-Induced Toxicity

In one aspect of the present disclosure, methods of reducing platinumdrug-induced toxicity in a cell expressing OCT2, including the steps ofproviding a platinum drug to the cell expressing OCT2, and providing anOCT2 inhibitor that reduces OCT2-mediated platinum drug uptake into thecell, where the OCT2 inhibitor reduces platinum drug-induced toxicity inthe cell are provided.

In another aspect, the present disclosure provides methods for reducingplatinum drug-induced toxicity in a subject, including the steps ofproviding a platinum drug to a subject that contains cells expressingOCT2, and providing an OCT2 inhibitor to the subject, where the OCT2inhibitor reduces OCT2-mediated platinum drug uptake into the cellsexpressing OCT2, thereby reducing platinum drug-induced toxicity in thesubject. In another aspect, the present disclosure provides methods forreducing platinum drug-induced toxicity in a subject, including the stepof providing an OCT2 inhibitor to a subject that contains cellsexpressing OCT2, where the subject has been or will be provided withplatinum drug and where the OCT2 inhibitor reduces OCT2-mediatedplatinum drug uptake into the cells expressing OCT2, thereby reducingplatinum drug-induced toxicity in the subject. In some embodiments, thesubject is a human. In other embodiments, the subject is a non-humananimal.

In another aspect, the present disclosure provides methods for reducingplatinum drug-induced neurotoxicity in a subject in need thereofcomprising administering a platinum drug that is oxaliplatin to thesubject in need thereof; and administering an effective dose of aselective Organic Cation Transporter 2 (OCT2) inhibitor to the subjectin need thereof, wherein the subject in need thereof has a cancer. Insome embodiments, the selective Organic Cation Transporter 2 (OCT2)inhibitor is not buflomedil. In some embodiments, the method furthercomprises a step of determining whether the cancer expresses at leastone of OCT1 or OCT3. In some embodiments, the cancer expresses at leastone of OCT 1 or OCT3.

In another aspect, the present disclosure provides methods for reducingplatinum drug-induced toxicity in a subject in need thereof comprisingadministering a platinum drug that is cisplatin to the subject in needthereof; and administering an effective dose of a selective OrganicCation Transporter 2 (OCT2) inhibitor to the subject in need thereof,wherein the toxicity is nephrotoxicity, ototoxicity or peripheralneuropathy, and wherein the subject in need thereof has a cancer. Insome embodiments, selective Organic Cation Transporter 2 (OCT2)inhibitor is not buflomedil. In some embodiments, the method furthercomprises a step of determining whether the cancer expresses at leastone of OCT1 or OCT3. In some embodiments, the cancer expresses at leastone of OCT 1 or OCT3.

In another aspect, the present disclosure provides methods for reducingplatinum drug-induced toxicity in a subject in need thereof comprisingdetermining an effective dose to achieve a plasma level of a selectiveOrganic Cation Transporter 2 (OCT2) inhibitor that is buflomedil or abuflomedil salt in the subject in need thereof, wherein the plasma levelis two to five times IC₅₀ of the selective Organic Cation Transporter 2(OCT2) inhibitor that is buflomedil or a buflomedil salt; administeringa platinum drug selected from the group consisting of oxaliplatin andcisplatin to the subject in need thereof; and administering an effectivedose of a selective Organic Cation Transporter 2 (OCT2) inhibitor thatis buflomedil or a buflomedil salt to the subject in need thereof,wherein the toxicity is nephrotoxicity, ototoxicity or peripheralneuropathy, and wherein the subject in need thereof has a cancer. Insome embodiments, the method further comprises a step of determiningwhether the cancer expresses at least one of OCT1 or OCT3. In someembodiments, the cancer expresses at least one of OCT 1 or OCT3.

In another aspect, the present disclosure provides methods for reducingplatinum drug-induced toxicity in a subject in need thereof comprisingadministering a selective Organic Cation Transporter 2 (OCT2) inhibitorwhich is selected from the group consisting of buflomedil, a buflomedilsalt, dolutegravir, and a dolutegravir salt, to the subject in needthereof, wherein the subject in need thereof has a cancer expressing atleast one of Organic Cation Transporter 1 (OCT1) or Organic CationTransporter 3 (OCT3), and the subject in need thereof has been or willbe administered with a platinum drug, such as cisplatin or oxaliplatin.In some embodiments, the method further comprises a step of determiningwhether the cancer expresses at least one of OCT1 or OCT3. In someembodiments the platinum drug is oxaliplatin and the platinumdrug-induced toxicity is neurotoxicity. In some embodiments the platinumdrug is cisplatin and the platinum drug-induced toxicity isneurotoxicity, nephrotoxicity or ototoxicity.

In some embodiments, the therapeutically effective amount of platinumdrug administered is known to cause platinum drug-induced toxicity insubjects. In some embodiments, the dose of selective OCT2 inhibitor iseffective to reduce platinum drug-induced toxicity in a subject in needthereof by at least 10% compared to platinum drug-induced toxicity insubjects treated with the same treatment protocol but withoutadministration of a selective OCT2 inhibitor.

Platinum Drug-Induced Toxicity

The methods of the present disclosure provide for a reduction ofplatinum drug-induced toxicity. Platinum drug-induced toxicity is aside-effect of treatment of cancer with platinum drugs. Whenadministered to subjects, platinum drugs typically have a therapeuticanticancer activity in tumor cells but may have an unwanted toxic effecton healthy cells. This unwanted platinum drug-induced toxicity ismediated, at least in part, by transport of platinum drugs into healthycells via OCT2.

Platinum drug-induced toxicity in a cell generally takes the form of acytotoxic effect on the cell. Reduction of platinum drug-inducedtoxicity in a cell may be measured by any relevant technique or assayknown to one of skill in the art. For example, toxicity in a cell may beassayed by assessing reduction in cell membrane integrity, which is acommon effect of cytotoxic compounds. One such assay, an LDH assay,measures the release of lactate dehydrogenase from a cell which isnormally sequestered inside the cell. Reduction of platinum drug-inducedtoxicity in a cell occurs when any amount of reduction occurs, asmeasured by the relevant assay. For example, toxicity in a cell may bereduced by at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, or atleast 90%.

Platinum drug-induced toxicity in a subject may manifest as toxicity indifferent physiological systems of the subject. Toxicity in a subjectcan be monitored by assessing parameters of general health of thesubject before and throughout the periods that the subject is providedwith OCT2 inhibitor and platinum drug. These parameters of generalhealth may include, for example, weight, hair loss, gait, skincondition, and loss of appetite and other gastrointestinal issues.Toxicity may also be assessed by measuring the prevalence or frequencyof toxicity among a group of subjects receiving the same platinum drugtreatment. A reduction in toxicity may be assessed by comparing theprevalence or frequency of toxicity in such a group of subjects overtime.

Platinum drug-induced toxicity may manifest as nephrotoxicity,neurotoxicity, hematoxicity, or ototoxicity. Nephrotoxicity may bemonitored with a blood test to measure creatinine clearance, serumcreatinine levels, or blood urea nitrogen levels. Decreased creatinineclearance, increased serum creatinine levels, and increased blood ureanitrogen levels indicate poor renal function. Neurotoxicity may bemonitored by assessing neurotoxicity symptoms, such as limb weakness ornumbness, loss of memory, vision, or intellect, uncontrollable obsessiveand/or compulsive behaviors, delusions, headache, cognitive andbehavioral problems and sexual dysfunction. In some embodiments wherethe platinum drug-induced toxicity is neurotoxicity, the neurotoxicityis peripheral neuropathy. In some embodiments, the peripheral neuropathyis Grade 3 or Grade 4 peripheral neuropathy. Ototoxicity may includecochleotoxicity characterized by high-frequency hearing loss andtinnitus. Ototoxicity may be analyzed by measuring auditory brainstemresponses. Chemotherapy-induced hematotoxicity is a complexmanifestation involving multiple mechanisms. It is however known in theart that reduction of neurotoxicity could also reduce hematotoxicity.For example, it is known that chemotherapy-induced nerve injury(neurotoxicity) with platinum derivatives produces a critical lesion inthe bone marrow, which impairs hematopoietic regeneration. Therefore, itis possible to indirectly shield hematopoietic cells from injury usingan agent that protects neuronal cells.

Reduction of platinum drug-induced toxicity in a subject occurs when anyamount of reduction occurs, as measured by the relevant assays or tests.In some embodiments, prevalence of platinum drug-induced toxicity in agroup of subjects is reduced by at least 10%, by at least 20%, by atleast 30%, by at least 40%, or by at least 50% as compared to theprevalence of platinum drug-induced toxicity in a group of subjects notprovided with an OCT2 inhibitor.

Platinum Drug-Induced Neurotoxicity

The methods of the present disclosure provide for a reduction ofplatinum drug-induced neurotoxicity. The methods of the presentdisclosure provide for a delay of onset of platinum drug-inducedneurotoxicity. In some embodiments, the neurotoxicity is Grade 0(normal), 1, 2, 3, or 4. In some embodiments, the delay is of onset ofGrade 3 or higher. In some embodiments, the delay is of onset of Grade 2or higher. In some embodiments, the neurotoxicity is damage to a motorneuron or sensory neuron. In some embodiments, the neurotoxicity isdamage to a motor neuron. In some embodiments, the neurotoxicity isdamage to a sensory neuron. In some embodiments, damage is to the DRG.

In some embodiments where the platinum drug-induced toxicity isneurotoxicity, the neurotoxicity is peripheral neurotoxicity. In someembodiments, the peripheral neurotoxicity is Grade 3 or Grade 4peripheral neuropathy. In some embodiments, the peripheral neurotoxicityis Grade 2 peripheral neuropathy.

Neurotoxicity can be assessed by NCI-CTCAE (National CancerInstitute-Common Terminology Criteria for Adverse Events), as shown inthe table below. In some embodiments, the neurotoxicity is assessed by amethod selected from National Cancer Institute-Common Toxicity Criteria(NCI-CTC) sensory scale, National Cancer Institute-Common ToxicityCriteria (NCI-CTC) motor scale, Total Neuropathy Score clinical version(TNSc) and European Organization for Research and Treatment of CancerCIPN specific self-report questionnaire (EORTC QOL-CIPN20).

Toxicity Grade 1 Grade 2 Grade 3 Grade 4 Grade 5 Cranial Asymptomatic,Moderate Severe Life- Death neuropathy detected on symptoms; symptoms;threatening (any) exam/testing only; limiting limiting self-consequences; intervention not instrumental care ADL urgent indicatedADL (activities activities of intervention of daily life) daily life)indicated Motor Asymptomatic, Moderate Severe Life- Death neuropathyclinical or symptoms; symptoms; threatening diagnostic limiting limitingself- consequences; observations only; instrumental care ADL urgentintervention not ADL activities activities of intervention indicated ofdaily life) daily life); indicated assistive device indicated SensoryAsymptomatic; Moderate Severe Life- Death neuropathy loss of deepsymptoms; symptoms; threatening tendon reflexes or limiting limitingself- consequences; paresthesia instrumental care ADL urgent ADLactivities activities of intervention of daily life) daily life)indicated Paresthesias Mild symptoms Moderate Severe symptoms; symptoms;limiting limiting self- instrumental care ADL ADL activities activitiesof of daily life) daily life)

In peripheral neuropathy, symptoms can range from numbness or tingling,to pricking sensations (paresthesia), or muscle weakness. Areas of thebody may become abnormally sensitive leading to an exaggeratedly intenseor distorted experience of touch (allodynia). In such cases, pain mayoccur in response to a stimulus that does not normally provoke pain.Severe symptoms may include burning pain (especially at night), musclewasting, paralysis, or organ or gland dysfunction. Damage to nerves thatsupply internal organs may impair digestion, sweating, sexual function,and urination. In the most extreme cases, breathing may becomedifficult, or organ failure may occur.

In some embodiments, the neurotoxicity is assessed by a measurementselected from sensory nerve action potential, sensory nerve conductionvelocity, cold pain threshold, heat pain threshold, mechanical painthreshold, cold detection threshold, warm detection threshold,mechanical detection threshold, vibration perception threshold, currentperception threshold, pinprick sensibility, deep tendon reflexes andgrip strength. These methods are described in Griffith, K. et. al.,Support Care Cancer 2014, 22(5): 1161-1169 and Chong, P S. and Cros, DP, Technology literature review: quantitative sensory testing, Muscle &nerve [0148-639X] Chong, Peter Siao Tick yr: 2004 vol: 29 issue: 5 pg:734-747.

In some embodiments where the platinum drug-induced toxicity isneurotoxicity, the neurotoxicity is chronic peripheral neuropathy. Incertain instances, in chronic peripheral neuropathy, the symptoms wouldhave been occurring for more than or about 2, 3, 4, 6, 7, 8, 9, 10, 11or 12 months. In some embodiments, the neurotoxicity is chronicneurotoxicity. In some embodiments, the neurotoxicity is acute syndrometransient neurotoxicity. In some embodiments, the neurotoxicity occurs 1hour to seven days after first treatment. In some embodiments, theneurotoxicity occurs 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19. 20, 21, 22, 23 hours after first treatment. In someembodiments, the neurotoxicity occurs 1, 2, 3, 4, 5, 6, or 7 days afterfirst treatment. In some embodiments, the neurotoxicity occurs after asubject completes treatment with a cumulative dose of at least 500mg/m². In some embodiments, the neurotoxicity occurs after a subjectcompletes treatment with a cumulative dose of at least 500, 600, 700, or800 mg/m².

Some peripheral neuropathies are due to damage to the axons, whileothers are due to damage to the myelin sheath, the fatty protein thatcoats and insulates the axon. Peripheral neuropathies may also be causedby a combination of both axonal damage and demyelination.Electrodiagnostic studies can help healthcare providers determine thetype of damage involved.

In some embodiments, neurotoxicity is damage to the dorsal rootganglion. A dorsal root ganglion (or spinal ganglion) (also known as aposterior root ganglion), is a cluster of nerve cell bodies (a ganglion)in a posterior root of a spinal nerve. The dorsal root ganglia containthe cell bodies of sensory (afferent) neurons.

Forms of Peripheral Neuropathy

Oxaliplatin “induces two clinically distinct forms of peripheralneuropathy; the neuromyotonia-like, acute, transient syndromecharacterized by cold-induced distal or perioral paresthesias andpharyngolaryngeal dysesthesias and the chronic form that, in most cases,is a pure sensory, axonal neuropathy with a stocking-and-glovedistribution.” (Argyriou, A., et. al., Cancer January 2013, 438-444).Cisplatin predominantly induces chronic peripheral neuropathy. The formsof chronic peripheral neuropathy are similar for oxaliplatin andcisplatin.

Acute OXAIPN is a transient peripheral nerve hyperexcitability syndromethat occurs shortly after the infusion of oxaliplatin. This form ofneuropathy usually occurs with low total cumulative doses and can betriggered or exacerbated by exposure to cold stimuli. Patients typicallyexperience paresthesia and dysesthesia of the hands and feet, as well asof the larynx and jaw. These symptoms tend to occur within hours ofexposure and are reversible over time, especially over the next fewhours and days.

Chronic OXAIPN occurs mainly in extremities and present with symptomssimilar to those of cisplatin-induced neuropathy. The development ofchronic OXAIPN is reported to correlate with the cumulative oxaliplatindose. A loss of sensation, dysesthesia, and even functional impairmentcan develop progressively after several cycles of oxaliplatin-basedtherapy. These effects are usually reversible over time, but might lastfor several months and have had a significant impact on the continuationof oxaliplatin-based treatment, as these painful symptoms often disruptthe chemotherapy schedule.

Providing a Platinum Drug

In some aspects of methods of the present disclosure, a platinum drug isprovided to a cell expressing OCT2 or a platinum drug is provided to asubject comprising cells expressing OCT2. The platinum drug may be anycompound that contains a platinum (II) or (IV) center, two substitutedor non-substituted cis-amines and two leaving groups. In someembodiments, the platinum drug is oxaliplatin. In some embodiments, theplatinum drug is cisplatin. In some embodiments, the platinum drug istetraplatin.

The cell or cells expressing OCT2 may include, for example, a kidneycell, a neuron cell, a sensory neuron cell, an ear cell, or a bloodcell. OCT2 expression in a cell may be assessed by any method founduseful by one of skill in the art, including assays measuringtranscription of the oct2 gene in the cell, assays measuring thepresence of OCT2 protein in the cell, and analysis of published datacontaining such measurements for various cell types.

The platinum drug may be provided to the cell expressing OCT2 or to thesubject comprising cells expressing OCT2 by any methods typicallycarried out in the lab or in a health care setting to administer drugsto a cell or to a subject. For example, cells may be incubated with theplatinum drug, or cells may be provided the platinum drug via the samemethods that are used to administer drugs to a subject. The platinumdrug may be provided to the subject, for example, enterally,intravenously, intramuscularly, intraperitoneally, orally, orparenterally.

In some embodiments, the amount of platinum drug provided to the subjectduring one treatment session is greater than what is provided understandard clinical practices. For example, a typical dose of oxaliplatinthat is provided under standard clinical practices is 60-130 mg/m² pertreatment cycle. In some embodiments, the cumulative amount of platinumdrug provided to the subject over the entire course of treatment isgreater than what is provided under standard clinical practices. Forexample, a typical cumulative dose of oxaliplatin over the entire courseof treatment under standard clinical practices is 780-850 mg/m², or, inrare cases, >1000 mg/m². In some embodiments, the platinum drug isprovided at a greater frequency than under standard clinical practices.For example, under standard clinical practices oxaliplatin is typicallyprovided to the subject every two or three weeks for 8-12 cycles over 6months.

Providing an OCT2 Inhibitor

In some aspects of methods of the present disclosure, an OCT2 inhibitoris provided that reduces OCT2-mediated platinum drug uptake into thecell expressing OCT2. OCT2-mediated platinum drug uptake into a cellexpressing OCT2 may be measured by any method deemed appropriate by oneof skill in the art. For example, the methods described in Examples 1and 2 below may be used. In some embodiments, OCT2-mediated platinumdrug uptake into the cell expressing OCT2 is inhibited by at least 50percent, at least 60 percent, at least 70 percent, at least 80 percent,or at least 90 percent as compared to OCT2-mediated platinum drug uptakeinto a cell not in the presence of the OCT2 inhibitor.

In some aspects of the methods of the present disclosure, an OCT2inhibitor is provided to the subject. In some embodiments, the OCT2inhibitor is buflomedil or a buflomedil salt. In other embodiments, theOCT2 inhibitor comprises imidazole. In some embodiments, the OCT2inhibitor is miconazole or a salt thereof. In some embodiments, the OCT2inhibitor is dolutegravir or a salt thereof.

The OCT2 inhibitor may be provided to the subject, for example,enterally, intravenously, intramuscularly, intraperitoneally, orally, orparenterally. The amount of OCT2 inhibitor provided varies but istypically just enough in order to reduce platinum drug-induced toxicity.In some embodiments, the amount of OCT2 inhibitor provided to thesubject is at a less than therapeutically effective dosage. With respectto the OCT2 inhibitor, a therapeutically effective dosage is a dosagethat is effective to achieve a desired therapeutic or prophylacticresult other than reduction of platinum drug-induced toxicity. Forexample, buflomedil is typically used to treat claudication or thesymptoms of peripheral arterial disease. Accordingly, for example, insome embodiments, the amount of OCT2 inhibitor provided to the subjectis at a dosage less than that could be therapeutically effective fortreatment of claudication. In some embodiments, the amount of OCT2inhibitor provided to the subject is 10 mg to 2000 mg per day. In someembodiments where the OCT2 inhibitor is buflomedil or a buflomedil salt,the amount of buflomedil or buflomedil salt provided to the subject is150 mg to 900 mg per day. In some embodiments where the OCT2 inhibitoris miconazole or a miconazole salt, the amount of miconazole ormiconazole salt provided to the subject is up to 2000 mg per day. Insome embodiments where the OCT2 inhibitor is dolutegravir or adolutegravir salt, the amount of dolutegravir or dolutegravir saltprovided to the subject is 10 mg to 200 mg per day.

The platinum drug and the OCT2 inhibitor may be provided to the subjectin any order or with any amount of overlap. In some embodiments, theplatinum drug and the OCT2 inhibitor are provided at the same time. Insome embodiments, the OCT2 inhibitor is provided to the subject beforethe platinum drug. In some embodiments, the OCT2 inhibitor is providedto the subject after the platinum drug. Drug pharmacokinetics can bemodified by using different administration routines and/or differentformulations. For example, slow intravenous infusion using drip-infusionpump or similar methods can be used to maintain desirable plasmaconcentrations of OCT2 inhibitors, including miconazole, dolutegravir,and buflomedil, over the course of platinum drug infusion for thepurpose of reducing platinum drug-induced toxicities.

In embodiments of the present disclosure where the OCT2 inhibitorcomprises imidazole, the OCT2 inhibitor may have certain characteristicsand functional attributes. In some embodiments, the OCT2 inhibitor has aC_(max,u)/IC₅₀ or C_(max)/IC_(50,app) of at least 1. In someembodiments, the OCT2 inhibitor has a C_(max,u)/IC₅₀ orC_(max)/IC_(50,app) of at least 3. C_(max) and C_(max,u) denote themaximum total and unbound plasma concentration of a drug, respectively.IC₅₀ denotes the half maximal inhibitory concentration assessed in vitroin protein-free assay buffer. IC_(50,app) denotes the half maximalinhibitory concentration assessed in vitro in serum or assay buffercontaining serum binding protein(s) such as albumin.

In some embodiments, the OCT2 inhibitor is not toxic to the cell at itsclinical concentration. Toxicity of the OCT2 inhibitor to the cellrefers to any cytotoxic effect on the cell measurable by any methodknown to one of skill in the art. In some embodiments, the OCT2inhibitor does not reduce anti-cancer activity of the platinum drug atits clinical concentration by more than 20% as compared to anti-canceractivity of the platinum drug at its clinical concentration in theabsence of the OCT2 inhibitor. Anti-cancer activity refers to thechemotherapeutic effect of a platinum drug on cancer cells. Platinumdrugs typically generate crosslinks in cancer cell DNA leading toapoptosis and cell growth inhibition. Anti-cancer activity may bemeasured by, for example, cytotoxicity assays on cancer cells.

In some embodiments, the OCT2 inhibitor does not reduce uptake of theplatinum drug at its clinical concentration into the cell viatransporters other than OCT2 by more than 20% as compared to uptake ofthe platinum drug at its clinical concentration via transporters otherthan OCT2 in the absence of the OCT2 inhibitor. In some embodiments, theOCT2 inhibitor does not reduce the efflux of the platinum drug at itsclinical concentration by more than 20% as compared to the efflux of theplatinum drug at its clinical concentration in the absence of the OCT2inhibitor. Uptake of the platinum drug into the cell via transportersother than OCT2 and efflux of the platinum drug may be measured by anyassay deemed suitable by one of skill in the art, including, forexample, the in vitro transporter assays described in Examples 3 and 4.“Clinical concentration” refers to a drug's plasma or serumconcentration when the drug is dosed at a clinically relevant range,which is below its maximum tolerated dose (MTD).

In some embodiments, the OCT2 inhibitor has a mean half-life that isgreater than 2 hours. Half-life refers to the amount of time it takesfor a substance, such as a drug or other molecule, to lose one-half ofits pharmacologic, physiologic, or radiological activity. The half-lifemay also describe the time that it takes for the blood plasmaconcentration of a substance to decrease by half.

Methods for Treating Cancer and for Increasing Efficiency of PlatinumDrug Treatment

In one aspect, the present disclosure provides methods for treatingcancer in a subject including the steps of providing a therapeuticallyeffective amount of platinum drug to the subject having cancer, wherethe subject comprises a cancerous cell expressing platinum drug uptaketransporters not inhibited by an Organic Cation Transporter 2 (OCT2)inhibitor, and providing an OCT2 inhibitor to the subject, therebytreating cancer in the subject. In some embodiments, the cancer is atype of cancer that is known to those skilled in the art to be amenableto treatment with/often treated with platinum drugs in standardtreatment protocols, including platinum drugs such as oxaliplatin and/orcisplatin. In some embodiments, the OCT2 inhibitor is buflomedil or abuflomedil salt. In other embodiments, the OCT2 inhibitor containsimidazole. In some embodiments, the OCT2 inhibitor is miconazole or asalt thereof. In some embodiments, the OCT2 inhibitor is dolutegravir ora salt thereof. In some embodiments the subject is a human. In otherembodiments, the subject is a non-human animal. In some embodiments, thesubject has a cancer who is or will be undergoing cancer treatment thatincludes administration of a platinum drug, such as oxaliplatin orcisplatin. In some embodiments, the OCT2 inhibitor is not buflomedil ora buflomedil salt.

In another aspect, the present disclosure provides methods for treatingcancer in a subject in need thereof comprising administering atherapeutically effective amount of a platinum drug that is oxaliplatinto the subject in need thereof; and administering an effective dose of aselective Organic Cation Transporter 2 (OCT2) inhibitor to the subjectin need thereof. In some embodiments, the selective Organic CationTransporter 2 (OCT2) inhibitor is not buflomedil. In some embodiments,the method further comprises a step of determining whether the cancerexpresses at least one of OCT1 or OCT3. In some embodiments, the cancerexpresses at least one of OCT 1 or OCT3.

In another aspect, the present disclosure provides methods for treatingcancer in a subject in need thereof comprising administering atherapeutically effective amount of a platinum drug that is cisplatin tothe subject in need thereof; and administering an effective dose of aselective Organic Cation Transporter 2 (OCT2) inhibitor to the subjectin need thereof. In some embodiments, the selective Organic CationTransporter 2 (OCT2) inhibitor is not buflomedil. In some embodiments,the method further comprises a step of determining whether the cancerexpresses at least one of OCT1 or OCT3. In some embodiments, the cancerexpresses at least one of OCT 1 or OCT3.

In another aspect, the present disclosure provides methods for treatingcancer in a subject in need thereof comprising determining an effectivedose to achieve a plasma level of a selective Organic Cation Transporter2 (OCT2) inhibitor that is buflomedil or a buflomedil salt in thesubject in need thereof, wherein the plasma level is two to five timesthe IC₅₀ of the selective Organic Cation Transporter 2 (OCT2) inhibitorthat is buflomedil or a buflomedil salt; administering a therapeuticallyeffective amount of a platinum drug selected from the group consistingof oxaliplatin and cisplatin to the subject in need thereof; andadministering an effective dose of a selective Organic CationTransporter 2 (OCT2) inhibitor that is buflomedil or a buflomedil saltto the subject in need thereof. In some embodiments, the methods furthercomprise a step of determining whether the cancer expresses at least oneof OCT1 or OCT3. In some embodiments, the cancer expresses at least oneof OCT 1 or OCT3.

Treating cancer refers to clinical intervention in an attempt to alterthe natural course of the subject or cell being treated during thecourse of clinical pathology. Desirable effects of treatment include,but are not limited to, decreasing the rate of disease progression,ameliorating or palliating the disease state, and remission or improvedprognosis. For example, an individual is successfully “treated” if oneor more symptoms associated with cancer are mitigated or eliminated,including, but not limited to, reducing the proliferation of (ordestroying) cancerous cells, decreasing symptoms resulting from thedisease, increasing the quality of life of those suffering from thedisease, decreasing the dose of other medications required to treat thedisease, and/or prolonging survival of subjects.

In another aspect, the present disclosure provides methods forincreasing efficacy of platinum drug treatment in a subject includingthe steps of providing a therapeutically effective amount of platinumdrug to the subject having cancer, where the subject comprises acancerous cell expressing platinum drug uptake transporters notinhibited by an OCT2 inhibitor; and providing an OCT2 inhibitor to thesubject, thereby increasing efficacy of the platinum drug treatment. Insome embodiments, the OCT2 inhibitor is buflomedil or a buflomedil salt.In other embodiments, the OCT2 inhibitor contains imidazole. In someembodiments, the OCT2 inhibitor is miconazole or a salt thereof. In someembodiments, the OCT2 inhibitor is dolutegravir or a salt thereof.

Efficacy of platinum drug treatment refers to the ability of theplatinum drug treatment to have the desired chemotherapeutic effect inthe subject. Efficacy is typically assessed by progression free survival(PFS), which is the length of time during or after treatment that apatient lives with a disease but it does not get worse, or by overallsurvival (OS), which is the length of time from either the date ofdiagnosis or the start of treatment for a disease that the patient withthe disease is still alive.

The platinum drug and the OCT2 inhibitor may be provided to the subjectin any order or with any amount of overlap. In some embodiments, theplatinum drug and the OCT2 inhibitor are provided at the same time. Insome embodiments, the OCT2 inhibitor is provided to the subject beforethe platinum drug. In some embodiments, the OCT2 inhibitor is providedto the subject after the platinum drug. Drug pharmacokinetics can bemodified by using different administration routines and/or differentformulations. For example, slow intravenous infusion using drip-infusionpump or similar methods can be used to maintain desirable plasmaconcentrations of OCT2 inhibitors, including miconazole, dolutegravir,and buflomedil, over the course of platinum drug infusion for thepurpose of treating cancer in the subject or increasing efficacy ofplatinum drug treatment in a subject.

The OCT2 inhibitor may be provided to the subject, for example,enterally, intravenously, intramuscularly, intraperitoneally, orally, orparenterally. The amount of OCT2 inhibitor provided varies but istypically just enough in order to increase efficacy of platinum drugtreatment in the subject. In some embodiments, the amount of OCT2inhibitor provided to the subject is at a less than therapeuticallyeffective dosage. With respect to the OCT2 inhibitor, a therapeuticallyeffective dosage is a dosage that is effective to achieve a desiredtherapeutic or prophylactic result other than treatment of cancer orincreasing efficacy of platinum drug treatment in the subject. Forexample, buflomedil is typically used to treat claudication or thesymptoms of peripheral arterial disease. Accordingly, for example, insome embodiments, the amount of OCT2 inhibitor provided to the subjectis at a dosage less than that could be therapeutically effective fortreatment of claudication. In some embodiments, the amount of OCT2inhibitor provided to the subject is 10 mg to 2000 mg per day.

The platinum drug may be any compound that contains a square-planarplatinum (11) or (IV) center, two substituted or non-substitutedcis-amines and two leaving groups. In some embodiments, the platinumdrug is oxaliplatin. In some embodiments, the platinum drug iscisplatin. In some embodiments, the platinum drug is tetraplatin. Theplatinum drug may be provided to the subject, for example, enterally,intravenously, intramuscularly, intraperitoneally, orally, orparenterally.

A therapeutically effective amount of a platinum drug refers to anamount of the drug that is effective to achieve a desired therapeutic orprophylactic result. Typically, the desired therapeutic result inproviding platinum drug to a subject is toxicity to cancer cells in thesubject. In some embodiments, the amount of platinum drug provided tothe subject during one treatment session is greater than what isprovided under standard clinical practices. For example, a typical doseof oxaliplatin that is provided under standard clinical practices is60-130 mg/m² per treatment cycle. In some embodiments, the cumulativeamount of platinum drug provided to the subject over the entire courseof treatment is greater than what is provided under standard clinicalpractices. For example, a typical cumulative dose of oxaliplatin overthe entire course of treatment under standard clinical practices is780-850 mg/m², or, in rare cases, >1000 mg/m². In some embodiments, theplatinum drug is provided at a greater frequency than under standardclinical practices. For example, under standard clinical practicesoxaliplatin is typically provided to the subject every two or threeweeks for 8-12 cycles over 6 months.

In some embodiments, the methods further comprise a step of providing atherapeutically effective amount of one or more additionalchemotherapeutic agents to the subject in addition to the platinum drug.The one or more additional chemotherapeutic agents may be, for example,5-fluorouracil, bevacizumab, capecetabine, gemcitabine, irinotecan, andleucovorin.

Types of Cancer

The methods of the present disclosure for treating cancer and forincreasing efficacy of platinum drug treatment may be used in subjectshaving any type of cancer that responds to platinum drug treatment. Forexample, the cancer may be adenocarcinoma of the pancreas, ampullary andperiampullary carcinoma, hepatocellular carcinoma, adenocarcinoma of theanus, appendiceal carcinoma, carcinoma of the colon or rectum,epithelial ovarian carcinoma, fallopian tube carcinoma. primaryperitoneal cancer, esophageal or esophagogastric junction carcinoma,gastric carcinoma, small bowel carcinoma, testicular cancer,cholangiocarcinoma, pancreatic adenocarcinoma, carcinoma of unknownprimary origin, chronic lymphocytic leukemia/small lymphocytic lymphoma,non-Hodgkin's lymphoma, adult T-cell leukemia/lymphoma, AIDS-relatedB-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma,gastric MALT lymphoma, nongastric MALT lymphoma, mantle cell lymphoma,mycosis fungoides/Sezary syndrome, splenic marginal zone lymphoma,peripheral T cell lymphoma, primary cutaneous B-cell lymphoma, primarycutaneous anaplastic large cell lymphoma (ALCL), lung cancer, livercancer, and gallbladder cancer.

In some embodiments, the cancer may be adenocarcinoma of the pancreas,ampullary and periampullary carcinoma, hepatocellular carcinoma,adenocarcinoma of the anus, appendiceal carcinoma, carcinoma of thecolon or rectum, epithelial ovarian carcinoma, fallopian tube carcinoma.primary peritoneal cancer, esophageal or esophagogastric junctioncarcinoma, gastric carcinoma, small bowel carcinoma, testicular cancer,cholangiocarcinoma, pancreatic adenocarcinoma, carcinoma of unknownprimary origin, chronic lymphocytic leukemia/small lymphocytic lymphoma,non-Hodgkin's lymphoma, adult T-cell leukemia/lymphoma, AIDS-relatedB-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma,gastric MALT lymphoma, nongastric MALT lymphoma, mantle cell lymphoma,mycosis fungoides/Sezary syndrome, splenic marginal zone lymphoma,peripheral T cell lymphoma, primary cutaneous B-cell lymphoma, primarycutaneous anaplastic large cell lymphoma (ALCL), lung cancer, livercancer, head and neck cancer, prostate cancer, or smooth muscle cancer,or gallbladder cancer.

In certain embodiments, the cancer is carcinoma of the colon or rectum.In certain embodiments, the cancer is liver cancer. In certainembodiments, the cancer is lung cancer. In certain embodiments, thecancer is ovarian cancer. In certain embodiments, the cancer is head andneck cancer. In certain embodiments, the cancer is prostate cancer. Incertain embodiments, the cancer is lymphoma. In certain embodiments, thecancer is smooth muscle cancer.

In some embodiments, the cancer is treatable with oxaliplatin. In someembodiments, the cancer is treatable with cisplatin.

In some embodiments, the methods of the present disclosure for treatingcancer and for increasing efficacy of platinum drug treatment may beused in subjects having a cancer expressing at least one of OrganicCation Transporter 1 (OCT1) or Organic Cation Transporter 3 (OCT3). Insome embodiments, the cancer expresses OCT1. In some embodiments, thecancer expresses OCT3. In some embodiments, the cancer expresses OCT1and OCT3.

Platinum Drug Uptake Transporters

In methods of the present disclosure for treating cancer and forincreasing efficiency of platinum drug treatment, the subject comprisesa cancerous cell expressing platinum drug uptake transporters notinhibited by an OCT2 inhibitor. In some embodiments, the method furthercomprises the step of determining whether the cancerous cell expressesplatinum drug uptake transporters not inhibited by an OCT2 inhibitor.Whether or not a platinum drug uptake transporter is inhibited by anOCT2 inhibitor may be determined by assays known to one of skill in theart. The step of determining whether the cancerous cell expressesplatinum drug uptake transporters not inhibited by an OCT2 inhibitor mayalso be carried out by reviewing published gene and/or proteinexpression data from various cancer cell types. In some embodiments, theplatinum drug uptake transporters not inhibited by an OCT2 inhibitor areOrganic Cation Transporter 1 (OCT1) uptake transporters. In someembodiments, the platinum drug uptake transporters not inhibited by anOCT2 inhibitor are Organic Cation Transporter 3 (OCT3) uptaketransporters. In some embodiments, the platinum drug uptake transportersnot inhibited by an OCT2 inhibitor are Copper Transporter I (CTR1)uptake transporters.

Methods of Predicting Efficacy

In one aspect the present disclosure provides methods of predicting theefficacy of a platinum drug and Organic Cation Transporter 2 (OCT2)inhibitor therapy in a subject having cancer including the steps ofobtaining a sample comprising at least one cancerous cell; anddetermining whether the cancerous cell expresses platinum drug uptaketransporters not inhibited by an OCT2 inhibitor, where platinum drug andOCT2 inhibitor therapy is likely to be effective in a subject where thecancerous cell expresses platinum drug uptake transporters not inhibitedby an OCT2 inhibitor.

In another aspect the present disclosure provides methods of predictingthe efficacy of platinum drug and Organic Cation Transporter 2 (OCT2)inhibitor therapy in a subject having cancer including the steps ofobtaining a sample comprising at least one cancerous cell; anddetermining whether the cancerous cell expresses OCT2, where theplatinum drug and OCT2 inhibitor therapy is not likely to be effectivein a subject where the cancerous cell primarily expresses OCT2.

In another aspect, the present disclosure provides methods of predictingthe efficacy of a platinum drug and Organic Cation Transporter 2 (OCT2)inhibitor therapy in a subject having cancer comprising determiningwhether the cancer expresses platinum drug uptake transporters notinhibited by a selective OCT2 inhibitor, wherein platinum drug and theselective OCT2 inhibitor therapy is likely to be effective in a subjectwhere the cancerous cell expresses platinum drug uptake transporters notinhibited by a selective OCT2 inhibitor. In some embodiments, the uptaketransporter is OCT1 or OCT3.

In another aspect, the present disclosure provides methods of predictingthe efficacy of platinum drug and Organic Cation Transporter 2 (OCT2)inhibitor therapy in a subject having cancer comprising determiningwhether the cancerous cell expresses OCT2, wherein the platinum drug anda selective OCT2 inhibitor therapy is not likely to be effective in asubject where the cancer primarily expresses OCT2. In some embodiments,the uptake transporter is OCT1 or OCT3.

Predicting the efficacy of platinum drug and selective OCT2 inhibitortherapy refers to the likelihood that the platinum drug and selectiveOCT2 inhibitor therapy will have the desired chemotherapeutic effect inthe subject. Efficacy is typically assessed by progression free survival(PFS), which is the length of time during or after treatment that apatient lives with a disease but it does not get worse, or by overallsurvival (OS), which is the length of time from either the date ofdiagnosis or the start of treatment for a disease that the patient withthe disease is still alive.

The methods of predicting efficacy as disclosed herein may be used in asubject having any type of cancer. In some embodiments, the platinumdrug is oxaliplatin. In some embodiments, the platinum drug iscisplatin. In some embodiments, the platinum drug is tetraplatin. Insome embodiments, the selective OCT2 inhibitor therapy includesadministering buflomedil or a buflomedil salt. In some embodiments, theselective OCT2 inhibitor therapy includes administering an OCT2inhibitor comprising imidazole. In some embodiments, the selective OCT2inhibitor therapy includes administering miconazole or a salt thereof.In some embodiments, the OCT2 inhibitor therapy includes administeringdolutegravir or a dolutegravir salt.

Obtaining Samples

The methods of predicting efficacy as described herein include the stepof obtaining a sample comprising at least one cancerous cell. Samplesmay be obtained by any method known in the art, including biopsy,aspiration, surgery, or sampling of a bodily fluid from the subject suchas blood, cerebral spinal fluid, amniotic fluid, peritoneal fluid, orinterstitial fluid. Typically the sample is obtained from a diseasedtissue or organ. The at least one cancerous cell may be detected, forexample, by analyzing the sample under a microscope or by detecting theexpression or presence of certain cancer markers in the sample.

Determining Step

In one aspect the methods of predicting efficacy as described hereininclude the step of determining whether the cancerous cell expressesplatinum drug uptake transporters not inhibited by an OCT2 inhibitor.Determining whether the cancerous cell expresses a platinum drug uptaketransporter not inhibited by an OCT2 inhibitor may involve any assaysknown to one of skill in the art for detecting the presence of specificplatinum drug uptake transporters or their expression in a cell. Thestep of determining whether the cancerous cell expresses platinum druguptake transporters not inhibited by an OCT2 inhibitor may also becarried out by reviewing published gene and/or protein expression datafrom various cancer cell types. In some embodiments, the platinum druguptake transporters not inhibited by an OCT2 inhibitor are OrganicCation Transporter 1 (OCT1) uptake transporters. In some embodiments,the platinum drug uptake transporters not inhibited by an OCT2 inhibitorare Organic Cation Transporter 3 (OCT3) uptake transporters. In someembodiments, the platinum drug uptake transporters not inhibited by anOCT2 inhibitor are Copper Transporter I (CTR1) uptake transporters.

In one aspect the methods of predicting efficacy as described hereininclude the step of determining whether the cancerous cell expressesOCT2. Determining whether the cancerous cell expresses OCT2 may involveany assays known to one of skill in the art for detecting the presenceof OCT2 or its expression in a cell. The step of determining whether thecancerous cell expresses OCT2 may also be carried out by reviewingpublished gene and/or protein expression data from various cancer celltypes.

Screening of Compounds for Reducing Platinum Drug-Induced Toxicity

Ideal properties for a platinum protective agent, composition, and/orregimen include: (i) maximum reduction, prevention, mitigation, and/ordelay in onset of platinum-associated toxicities (and associatedtreatment interruptions, delays or dose modifications due to suchtoxicities); (ii) a lack of interference with anti-tumor activity andlack of tumor desensitization to cytotoxic effects of platinum therapy;(iii) a safety profile that is medically acceptable; (iv) exploitationof biochemical and pharmacological mechanisms to reduce, prevent,mitigate, and/or delay platinum-associated toxicity; and (v) increasesin chemotherapeutic index by allowing increases in dose, frequency,and/or duration of primary platinum treatment.

As used herein, a selective OCT2 inhibitor has an IC₅₀ for OCT2-mediatedtransport of 20 μM oxaliplatin in human serum or an assay solutioncontaining 4% bovine serum albumin, of less than or equal to 5 μM; andhas an IC₅₀ for OCT2-mediated transport of 20 μM oxaliplatin in humanserum or an assay solution containing 4% bovine serum albumin that is atleast 10-fold less than the selective OCT2 inhibitor IC₅₀ for 20 μMoxaliplatin transport in human serum or an assay solution containing 4%bovine serum albumin mediated by OCT-1, OCT-3, and MATE-1.

In some embodiments, a selective OCT2 inhibitor has an IC₅₀ forOCT2-mediated transport of 20 μM oxaliplatin in human serum or an assaysolution containing 4% bovine serum albumin, of less than or equal to 2,3, or 4 μM.

In some embodiments, a selective OCT2 inhibitor has an IC₅₀ forOCT2-mediated transport of 20 μM oxaliplatin in human serum or an assaysolution containing 4% bovine serum albumin that is at least 15, 20,25-fold less than the selective OCT2 inhibitor IC₅₀ for 20 μMoxaliplatin transport in human serum or an assay solution containing 4%bovine serum albumin mediated by OCT-1, OCT-3, and MATE-1.

In some embodiments, a selective OCT2 inhibitor has the one or more ofthe following additional criteria:

1) C_(max) under standard or current use is 2 times of OCT2 IC₅₀; and

2) maximum tolerated plasma concentration (MTC) is greater than 15 timesOCT2 IC₅₀, or 4 times of calculated OCT2 IC₉₀.

In some embodiments, another criterion is that the selective OCT2inhibitor does not reduce the efficacy of the platinum drug.

If a platinum-protective agent is capable of increasing the therapeuticindex of an active, but otherwise toxic, platinum drug, composition,and/or regimen, it may lead to significant benefit to the subject byincreasing tumor response rate, increasing time to tumor progression,and overall patient survival. Prevention of OXA-IPN includes complete orpartial prevention. If partial prevention is obtained, it should besufficient to observe some clinical improvement in presence of drug.Long term toxicity being related to platinum levels in DRG, preventioncould also mean a decrease in platinum uptake in DRG while keeping theuptake of OXA constant in the tumor. Another way to measure successfulprevention is to improve the ratio of uptake between tumor and DRG(Tumor/DRG) and keeping that ratio above its original value measured inabsence of drug.

OCTs the transporters involved in the uptake of oxaliplatin, with OCT2being responsible for the uptake of oxaliplatin into DRG and kidneycells whereas OCT1 is mostly responsible for the uptake of oxaliplatininto tumor cells. A ratio of blockage called DRG/tumor by calculatingthe ratio OCT2/OCT1 uptake ratio can be established and is useful forscreening.

A large series of compounds were screened to find compounds that blocksOCT2-mediated, but not OCT1-mediated, uptake of oxaliplatin in cells(and thus are selective OCT2 inhibitors). Preferably, the compoundstested were studied for their DRG/tumor ratio expressed in terms ofOCT2/OCT1 binding ratio.

A particularity of the OCT2 transporter has been described in theliterature by Belzer (Belzer, M., et. al, J Pharmacol Exp Ther. 2013August; 346(2): 300-310). Indeed OCT2 is known to have several bindingsites and therefore inhibitors for the OCT2 transporters are known to besubstrate dependent. Belzer's observations were tested by comparing theeffect of several inhibitors on a selection of substrates. Consistentwith the results from Belzer, a compound's inhibitory potency ofOCT2-mediated transport varies significantly depending on the OCT2substrates being investigated. Therefore, a skilled artisan should notrely on the published OCT2 inhibitory values unless oxaliplatin was usedas a substrate.

Platinum Drugs

Platinum drugs are compounds that contain a platinum (II) or (IV)center, two substituted or non-substituted cis-amines and two leavinggroups. They form highly reactive, charged, platinum complexes whichbind to nucleophilic groups in DNA, inducing intra- and inter-strand DNAcrosslinks resulting in apoptosis and cell growth inhibition. Commonlyused platinum drugs include cisplatin (structure: square-planar platinum(II) center, containing two cis-amines and two chloride groups),carboplatin (structure: square-planar platinum (II) center, containingtwo cis-amines and bidentate dicarboxylate), and oxaliplatin (structure:square-planar platinum (II) center, bidentate ligand1,2-diaminocyclohexane and a bidentate oxalate group). Additionalplatinum drugs include lipoplatin, nedaplatin, eptaplatin (heptaplatin),picoplatin, satraplatin, tetraplatin, iproplatin, lobaplatin, andtriplatin.

Structures of platinum drugs include, for example,

Several of these platinum drugs are drugs on various world markets.Others are drug candidates currently undergoing human clinical trials.In some embodiments, the platinum drug is one that has resulted inintolerable levels of toxicity when administered in clinical trials. Forexample, tetraplatin (ormaplatin) failed in human clinical trials due toan intolerable level of neurotoxicity. Anti-cancer therapy with suchdrugs would greatly benefit from the methods disclosed herein.

In some embodiments, oxaliplatin is provided as a powder or a solution.Oxaliplatin may be administered as an intravenous infusion or injection.Oxaliplatin can be obtained from commercial sources (Ranbaxy Limited,Mumbai, India; Accord Healthcare Limited, Durham, N.C.; Actavis,Parsippany-Troy Hills, N.J.; and Hospira, San Jose, Calif.).

In some embodiments, cisplatin is provided as a solution. Cisplatin maybe administered as an intravenous infusion. Cisplatin can be obtainedfrom commercial sources (Accord Healthcare Limited, Durham, N.C.;Hospira, San Jose, Calif.; and Sandoz, Holzkirchen, Upper Bavaria,Germany).

Uptake of platinum drugs by transporters typically involves uptake ofthe metabolites of the drug. Platinum drugs are metabolized and thenthese metabolites are transported into cells. For example, OCT2 maytransport cisplatin analog(s) more efficiently than transportingcisplatin itself.

OCT2 Inhibitors

In some embodiments, the selective OCT2 inhibitor is buflomedil or abuflomedil salt. The structure of buflomedil is

Its IUPAC name is4-pyrrolidin-1-yl-1-(2,4,6-trimethoxyphenyl)butan-1-one. In someembodiments, buflomedil is provided as a solution. Buflomedil may beadministered orally or intravenously. Buflomedil can be obtained fromcommercial sources (Loftyl™, Abbott, Chicago, Ill.).

Buflomedil is a vasoactive drug and has been used to treat claudicationor symptoms of peripheral arterial disease. The standard of care forbuflomedil is 300 mg orally per day with a maximum of 600 mg per day; orintravenous bolus of 200 mg maximum per day for a patient. The MTC(maximum tolerable concentration) is 10 mg/l.

In some embodiments, the OCT2 inhibitor comprises imidazole. Imidazolesare organic compounds with the formula (CH)2N(NH)CH, are planarheterocyclic (five-member ring), and are water soluble. Imidazoles arepart of the class of azole antifungal drugs. They include, for example,bifonazole, butoconazole, clotrimazole, econazole, fenticonazole,isoconazole, ketoconazole, luliconazole, miconazole, omoconazole,oxiconazole, sertaconazole, sulconazole, tioconazole, imidazoleantibiotics (Nitroimidazoles) such as 6-Amino PA824, azanidazole,benzonidazol, dimetridazole, megazol, metronidazole, nimorazole,ornidazole, tinidazole, and the imidazole sedative, midazolam. In someembodiments, the selective OCT2 inhibitor is a salt of an imidazole.

In some embodiments, imidazole contains the following structure:

R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 X miconazole H H Cl H Cl Cl H Cl H H Osulconazole H H Cl H H Cl H Cl H H S econazole H H Cl H H Cl H Cl H H Oisoconazole Cl H H H Cl Cl H Cl H H O

In some embodiments, imidazole contains the following structure:

R1 R2 R3 R4 R5 X Y sertaconazole Cl H Cl H H O 7-chloro-1-benzothiopheneFenticonazole Cl H Cl H H O Phenyl-sulfanyl phenyl thioconazole Cl H ClH H O 2-chloro-3-thienyl

In some embodiments, the selective OCT2 inhibitor is dolutegravir or asalt of dolutegravir. The structure of dolutegravir is

In some embodiments, dolutegravir (Trivicay™, Viiv Healthcare, UK) isprovided as a tablet. Dolutegravir may be administered orally.Dolutegravir can be obtained from commercial sources (Trivicay™, ViivHealthcare, UK).

Effective Dose of Selective OCT2 Inhibitor

An effective dose of a selective OCT2 inhibitor refers to an amount of aselective OCT2 inhibitor that can sufficiently reduce OCT2 transport ofa platinum drug into a cell. In some embodiments, the amount of aselective OCT2 inhibitor can completely block transport of a platinumdrug into a cell or block 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85,80, 75, or 70% of transport of a platinum drug into a cell. In someembodiments, the platinum drug which is a substrate for thedetermination is oxaliplatin. Measurement is performed by comparingplatinum accumulation in cells that express OCT2 and without OCT2expression (control cells). A 100% blockage means platinum amount in theOCT2 cells and control cells are the same

In some embodiments, the dose of selective OCT2 inhibitor is effectiveto reduce platinum drug-induced toxicity in said subject in need thereofby at least 10% compared to platinum drug-induced toxicity in subjectstreated with the same treatment protocol but without administration of aselective OCT2 inhibitor.

In some embodiments, the dose of selective OCT2 inhibitor is effectiveto minimize platinum drug-induced neurotoxicity in said subject in needthereof.

In some embodiments, the dose of selective OCT2 inhibitor is effectiveto prevent platinum drug-induced neurotoxicity of Grade 3 or higher, asassessed by a method selected from the group consisting of NationalCancer Institute-Common Toxicity Criteria (NCI-CTC) sensory scale,National Cancer Institute-Common Toxicity Criteria (NCI-CTC) motorscale, Total Neuropathy Score clinical version (TNSc), and EuropeanOrganization for Research and Treatment of Cancer CIPN specificself-report questionnaire (EORTC QOL-CIPN20), in a patient treated witha cumulative dose of at least 100 mg/m². In some embodiments, thecumulative dose at least 100 mg/m², 200 mg/m², 300 mg/m², 400 mg/m² or500 mg/m². In some embodiments, the dose of selective OCT2 inhibitor iseffective to prevent platinum drug-induced neurotoxicity of Grade 2 orhigher. In some embodiments, the prevention is a delay of onset or delayof promotion to the next grade of neurotoxicity.

In some embodiments, the dose of selective OCT2 inhibitor is effectiveto prevent platinum drug-induced neurotoxicity of Grade 3 or higher, asassessed by a method selected from National Cancer Institute-CommonToxicity Criteria (NCI-CTC) sensory scale, National CancerInstitute-Common Toxicity Criteria (NCI-CTC) motor scale, TotalNeuropathy Score clinical version (TNSc) and European Organization forResearch and Treatment of Cancer CIPN specific self-report questionnaire(EORTC QOL-CIPN20), in a patient treated with a platinum drug doseintensity of at least 30 mg/week/m². In some embodiments, the platinumdrug dose intensity is at least 30 mg/week/m², 40 mg/week/m², 50mg/week/m² or 60 mg/week/m². In some embodiments, the platinum drug doseintensity is at least 30 mg/week/m² to 50 mg/week/m². In someembodiments, the dose of selective OCT2 inhibitor is effective toprevent platinum drug-induced neurotoxicity of Grade 2 or higher. Insome embodiments, the prevention is a delay of onset or delay ofpromotion to the next grade of neurotoxicity.

In some embodiments, the dose of selective OCT2 inhibitor is effectiveto prevent platinum drug-induced neurotoxicity of Grade 3 or higher, asassessed by a method selected from National Cancer Institute-CommonToxicity Criteria (NCI-CTC) sensory scale, National CancerInstitute-Common Toxicity Criteria (NCI-CTC) motor scale, TotalNeuropathy Score clinical version (TNSc) and European Organization forResearch and Treatment of Cancer CIPN specific self-report questionnaire(EORTC QOL-CIPN20), in a patient treated with a platinum drug dose of atleast 80 mg/m². In some embodiments, the platinum drug dose is at least80 mg/m², 90 mg/m², 100 mg/m², 110 mg/m², 120 mg/m², 130 mg/m², or 140mg/m². In some embodiments, the dose of selective OCT2 inhibitor iseffective to prevent platinum drug-induced neurotoxicity of Grade 2 orhigher. In some embodiments, the prevention is a delay of onset or delayof promotion to the next grade of neurotoxicity.

Prevention, as used herein, can refer to any action providing a benefitto a subject at risk of being afflicted with a condition or disease,including avoidance of the development of the condition or disease or adecrease of one or more symptoms of the condition or disease. Withregard to prevention of platinum drug-induced neurotoxicity of a certaingrade, the promotion or progression to the next grade for the subject isavoided.

In some embodiments, the selective OCT2 inhibitor is administered in adose effective to reduce neurotoxicity. Neurotoxicity can result insensitivities to touching cold items, difficulty swallowing coldliquids, throat discomfort, and muscle cramps. In some embodiments, theneurotoxicity is Grade 0, 1, 2, 3, or 4. In some embodiments, theneurotoxicity is damage to a motor neuron or sensory neuron. In someembodiments, the selective OCT2 inhibitor is administered in a doseeffective to minimize damage to dorsal root ganglia (DRG).

In some embodiments, neurotoxicity is assessed by determining nerveconduction. In some embodiments, the nerve conduction study is conductedin one of radial, dorsal sural, sural and ulnar nerves. These methodsare discussed in Velasco, R., et. al, J Neurol Neurosyrg Psychiatry2014; 85:392-398.

In some embodiments, the nerve conduction study comprises measuringsensory nerve action potential (SNAP). In some embodiments, the nervecondition study comprises measuring nerve conduction velocity (NCV). Insome embodiments, neurotoxicity is assessed by determining sensitivityto cold temperature. In some embodiments, neurotoxicity is assessed bydetermining sensitivity to a mechanical stimulus.

In some embodiments, neurotoxicity is assessed by a test method selectedfrom thermal detection threshold, mechanical detection threshold,vibration perception threshold, current perception threshold, pinpricksensibility, deep tendon reflexes and grip strength. These methods arediscussed in Griffith, K. et. al., Support Care Cancer 2014, 22(5):1161-1169.

In some embodiments, neurotoxicity is assessed by National CancerInstitute-Common Toxicity Criteria (NCI-CTC) sensory scale. In someembodiments, neurotoxicity is assessed by Total Neuropathy Scoreclinical version (TNSc). In some embodiments, neurotoxicity is assessedby the European Organization for Research and Treatment of Cancer CIPNspecific self-report questionnaire (EORTC QOL-CIPN20). These methods arediscussed in Kauto et al. “Oxaliplatin and National CancerInstitute-Common Toxicity Criteria in the Assessment ofChemotherapy-induced Peripheral Neuropathy” Anticancer Research 31:3493-3496 (2011); and Cavaletti, G., et. al., European Journal of Cancer46 (2010) 479494.

Time-Concentration Profile of a Selective OCT2 Inhibitor Under ClinicalSettings

Clinically, platinum-based anti-cancer agents, including oxaliplatin andcisplatin, are administrated via intravenous infusion over a period oftime (typically 2-4 hours, T_(PD) in Figure A) in a treatment cycle,during which platinum drugs reach high plasma levels and are more likelyto cause adverse effects, including peripheral neuropathy andnephrotoxicity. Therefore, it is important that during the course ofplatinum drug administration (T_(PD)), the plasma level of a selectiveOCT2 inhibitor is maintained between two pre-determined levels (C_(L)and C_(H) in FIG. 33) in order to achieve desirable protective effectswhile at the same time, minimizing the adverse effects of the OCT2inhibitor itself. This therapeutic window is depicted in Figure A as thehatched area. Another way to describe such pharmacokinetic profile isthrough defining a preventive time period (T_(prev)) during which theplasma level of the OCT2 inhibitor is maintained between C_(L) andC_(H), and T_(prev) should cover the entire period (T_(PD)) of platinumdrug administration as depicted in Figure A. Outside of T_(prev), it isdesirable to keep inhibitor level as low as possible so as to minimizeits other pharmacological effects including adverse effects. Suchdesirable time-plasma level profile can be achieved through applyingcommonly practiced drug administration techniques such as oral,intravenous injection, intravenous infusion, or combination of such; andthrough optimizing the formulation of the selective OCT2 inhibitor suchas extended release.

It is important to note that the approach above for eliciting optimalprotective effects of a selective OCT2 inhibitor againstplatinum-drug-induced toxicities is in contrast to most othertherapeutic interventions whereas treatment efficacy of a drug isprimarily driven by its accumulative plasma exposure (drug plasma levelintegrated over time, typically 12-24 hours, which is commonly referredas area under the curve/AUC) [Benet, L. and Hoener, B., Clin PharmacolTher 2002; 71:115-21].

The desirable plasma concentration range of a selective OCT2 inhibitorcan be determined as follow: C_(L) can be determined based on theinhibitor's in vitro OCT2 IC₅₀ value, which is measured in 100% humanserum or assay buffer containing physiological level of serum albumin.Depending on the level of OCT2 inhibition during the course of platinumdrug administration, C_(L) may be set to a value that is not lower thanthe in vitro OCT2 IC₅₀ value, which corresponds to an estimated 50%reduction of OCT2 mediated transport of a platinum drug in vivo. Morepreferably, C_(L) may be set to two times or four times the in vitroOCT2 IC₅₀ value, which corresponds to estimated 70% and 90% OCT2inhibition, respectively. For example, for buflomedil with measured IC₅₀value of 1.4±0.2 uM (Table 4), the C_(L) may be set to 1.4 uM (0.43mg/l); or more preferably, 2.8 uM (0.86 mg/1) or 5.6 uM (1.72 mg/l) foreliciting more protective effects. In the case of dolutegravir, themeasured IC₅₀ value is 3.4±1.0 uM (Table 4), thus the C_(L) may be setto 1× (3.4 uM or 1.4 mg/1), 2× (6.8 uM or 2.8 mg/l) or 4× (13.6 uM or5.6 mg/l) the IC₅₀ value.

C_(H) can be determined based on clinical and preclinical toxicitystudies of a selective OCT2 inhibitor, such as the C_(max) of a drugunder its clinical maximum tolerated dose (MTD). For example, it is welldocumented that buflomedil overdose can lead to fatal cardio- andneurotoxicity [European Medicines Agency 2012, Bucolo 2012], whichcontributed to its marketing suspension in Europe and in a number ofother countries. It's documented that the serious toxicities areassociated with buflomedil plasma levels above 10 mg/l (32.5 uM)[European Medicines Agency 2012, Bourguignon 2012]. Hence, it iscritical to ensure that buflomedil plasma level stay below thetoxicity-triggering level by setting C_(H) to 10 mg/l or lower. In thecase of dolutegravir, a preclinical 14-week toxicity study in monkeyreported that at the NOAEL (no-observed-adverse-effect-level) dose, themean plasma C_(max) in females is 23.5 mg/l (54.8 uM) [PMDA 2012], thusC_(H) can be set to equal or to be below this value.

Dose Adjustment of a Selective OCT2 Inhibitor

The plasma level of a drug is affected by many factors, including butnot limited to: body weight, gender, age and function of major drugclearance organs such as kidney and liver function [FDA, Guidance forIndustry: Population Pharmacokinetics, 1999]. Therefore, significantvariabilities in drug plasma levels are often seen in subjects under thesame drug dose and route of administration. Such inter-individualvariabilities not only can influence the protective effects of aselective OCT2 inhibitor, but more importantly, could lead to seriousadverse effects when the inhibitor stays above its safe level C_(H).This is particularly important for drugs such as buflomedil because highplasma exposure is reported to cause severe toxicities and mortality[European Medicines Agency, Procedure number: EMEA/H/A-107/1293(http://www.ema.europa.eu/docs/en_GB/document_library/Referrals_document/Buflomedil_107/WC500128578.pdf)2012; Bucolo, C., et. al., pharmacoepidemiology and drug safety 2012;21: 1190-1196]. To demonstrate the need for dose adjustment, weconducted a pharmacokinetic simulation in two group of subjectsadministrated with 450 mg buflomedil through a constant rate intravenous(IV) infusion (150 mg/hour over 3 hours). Pharmacokinetic parametersused in the simulation are from two distinct (by weight, age and height)groups of patients, which exhibited significantly difference in averagebuflomedil clearance (Group A 292 ml/min vs. Group B 99.5 ml/min)[Gundert-Remy, U., et. al., Eur J Clin Pharmacol (1981) 20: 459-463;Bourguignon, L., et. al., Fundam Clin Pharmacol. 2012 April;26(2):279-85]. As shown in FIG. 34, despite the plasma levels ofbuflomedil of Group A subjects are in the desirable range defined byC_(L) and C_(H), however, in Group B subjects of seniors (average age 82yrs) with impaired renal clearance, the same dose results in dangerouslyhigh levels of buflomedil (>10 mg/l). The PK simulation also suggests anearly 70% dose reduction for the Group B subjects in order to achievesimilar plasma level of buflomedil in Group A subjects. The PKsimulation also demonstrates that IV infusion of buflomedil at aconstant rate results in slow change in its plasma level over time.Combining an IV bolus injection and an IV infusion, or using variablerate IV infusion, or using other methods described in [Shargel, L., et.al, Applied Biopharmaceutics and Pharmacokinetics, 6^(th), 2012] may beapplied in order to achieve more consistent plasma level of a selectiveOCT2 inhibitor during the course of administration.

Many approaches can be used to tailor drug dose for individuals [Klotz,U., J Clin Chem Clin Biochem. 1983 November; 21(11):649-58; Jelliffe,R., Ther Drug Monit. 2000 June; 22(3):325-9]. For example, a clinicalapproach may involve applying a low or normal dose to a subject,followed by measuring the plasma drug level, which can be used to guidedose adjustment for that subject as needed. As another example, doseadjustment can also be guided by population pharmacokinetics analysis[FDA, Guidance for Industry: Population Pharmacokinetics, 1999], whichis commonly practiced to identify measurable factors that are associatedwith variabilities in drug level. Such factors include, but are notlimited to, weight, body surface area, height, age, gender, alcohol use,smoking, life style, renal function (commonly assessed by creatinineclearance test), liver function (commonly assessed withIndocyanine-green clearance test), genetic polymorphism andco-medications.

Pharmacokinetic Profile of OCT2 Inhibitor Concentration

The present disclosure provides the disclosed methods, in which theselective OCT2 inhibitor is administered at a dose that results in itsplasma concentration during the period of platinum drug administrationis less than its maximum tolerated plasma concentration (MTC) andgreater than 1×, 2×, 3×, 4× of its IC₅₀ value for OCT2-mediatedtransport of 20 μM oxaliplatin assessed in human serum or an assaybuffer containing 4% bovine serum albumin.

In some embodiments, the dose of buflomedil or a buflomedil saltadministered in a subject is adjusted based on at least one of thefactors of the subject, including body weight, body surface area,height, age, gender, alcohol use, tobacco use, life style, renalfunction, liver function, genetic polymorphism and co-medications. Insome embodiments, the dose of buflomedil or a buflomedil salt results inits plasma level during the period of platinum administration at least0.43 mg/l, 0.86 mg/l, 1.29 mg/l, 1.72 mg/l or 2.15 mg/l. In someembodiments, the dose of buflomedil or a buflomedil salt is at least 50,75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500, 525, 550, 575, or 600 mg. In some embodiments, thedose of buflomedil or a buflomedil salt is at least 100 mg, 200 mg, 400mg or 600 mg. In some embodiments, the dose of buflomedil or abuflomedil salt is at least 300 mg, 450 mg, 600 mg, 800 mg or 1000 mg.In some embodiments, the dose of buflomedil or a buflomedil salt is atleast 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 8 mg/kg or10 mg/kg.

In some embodiments, the dose of dolutegravir or a dolutegravir saltresults in its plasma level during the period of platinum administrationat least 1.4 mg/l, 2.8 mg/l, 4.2 mg/l, 5.6 mg/l or 7.0 mg/l. In someembodiments, the dose of dolutegravir or a dolutegravir salt is at least50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg or 400 mg. In someembodiments, the dose of dolutegravir or a dolutegravir salt is at least50 mg, 75 mg, 100 mg, 150 mg, 200 mg or 300 mg. In some embodiments, thedose of dolutegravir or a dolutegravir salt is at least 1 mg/kg, 2mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg or 6 mg/kg.

In some embodiments, the dose adjustment is based on the characteristicsof the subject as described above with reference to the data chartsdescribed herein as development for individual drugs.

Pharmaceutical Compositions

In one aspect the present disclosure provides pharmaceuticalcompositions containing a platinum drug, an OCT2 inhibitor, and apharmaceutically acceptable carrier. In some embodiments, the OCT2inhibitor is buflomedil or a buflomedil salt. In other embodiments, theOCT2 inhibitor contains imidazole. In some embodiments, the OCT2inhibitor is miconazole or a salt thereof. In some embodiments, the OCT2inhibitor is dolutegravir or a salt thereof. In some embodiments, theplatinum drug is oxaliplatin or cisplatin. In some embodiments, theplatinum drug is oxaliplatin. In some embodiments, the platinum drug iscisplatin. In some embodiments, a pharmaceutical composition isformulated for intravenous administration comprising a platinum drugthat is oxaliplatin, a selective OCT2 inhibitor selected from the groupconsisting of buflomedil, a buflomedil salt, dolutegravir, and adolutegravir salt, and a pharmaceutically acceptable carrier.

In some embodiments, a pharmaceutical composition is formulated forintravenous administration comprising a platinum drug that isoxaliplatin or cisplatin, a selective OCT2 inhibitor selected from thegroup consisting of buflomedil, a buflomedil salt, dolutegravir, and adolutegravir salt, and a pharmaceutically acceptable carrier.

In some embodiments, a pharmaceutical composition is formulated forintravenous administration comprising a platinum drug that isoxaliplatin or cisplatin, a selective OCT2 inhibitor that is buflomedilor a buflomedil salt, and a pharmaceutically acceptable carrier.

In some embodiments, the platinum drug is oxaliplatin. In someembodiments, the platinum drug is cisplatin. In some embodiments, theplatinum drug is tetraplatin. In some embodiments, the amount ofplatinum drug is greater than what is present in a standardpharmaceutical composition containing a platinum drug. For example, astandard pharmaceutical composition containing oxaliplatin is usuallydosed as a powder to be reconstituted with water with the followingstrengths: 50 mg/vial, 100 mg/vial, or 200 mg/vial. A standardpharmaceutical composition containing cisplatin is usually provided as a1 mg/ml solution ready for injection in large vial containing 200 mg/200ml. Tetraplatin is not an approved drug but the injected amount isexpected to be similar to cisplatin (5 mg/kg) with a similarformulation.

In some embodiments, the pharmaceutical composition further contains atherapeutically effective amount of one or more additionalchemotherapeutic agents in addition to the platinum drug. The additionalchemotherapeutic agent may be, for example, 5-fluorouracil, bevacizumab,capecetabine, gemcitabine, irinotecan, or leucovorin.

Any pharmaceutically acceptable carrier may be used. In someembodiments, compositions of the present disclosure are formulated in aconventional manner using one or more physiologically acceptablecarriers including, e.g., excipients and auxiliaries which facilitateprocessing of the active compounds into preparations which are suitablefor pharmaceutical use. Suitable pharmaceutically acceptable carriersinclude, without limitation, physiological saline, aqueous buffersolutions, water for injection use, dextrose solution, solvents and/ordispersion media. The use of such carriers is well known in the art. Thecarrier is preferably sterile. In some embodiments, the carrier isstable under the conditions of manufacture and storage and preservedagainst the contaminating action of microorganisms, such as bacteria andfungi, through the use of, for example and without limitation, parabens,chlorobutanol, phenol, ascorbic acid, or thimerosal.

Examples of materials and solutions that can serve as pharmaceuticallyacceptable carriers include, without limitation: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

Proper formulation of the compositions of the present disclosure may bedependent upon the route of administration chosen. A summary ofpharmaceutical compositions described herein is found, for example, inRemington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton,Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975;Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms,Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms andDrug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).In some embodiments, the pharmaceutical composition is formulated forenteral, intravenous, intramuscular, intraperitoneal, oral, orparenteral administration. In some embodiments, the pharmaceuticalcomposition is formulated for intravenous administration. In certainembodiments, multiple compositions are provided and formulated fordifferent routes of administration.

Articles of Manufacture

The present disclosure relates to a kit comprising a platinum drug thatis oxaliplatin or cisplatin; a selective OCT2 inhibitor; andinstructions for use. The kit may further comprise instructions for use,e.g., for reducing platinum drug-induced neurotoxicity; treating cancer;or creasing patient compliance for treating cancer. The instructions foruse are generally written instructions, although electronic storagemedia (e.g., magnetic diskette or optical disk) containing instructionsare also acceptable.

The present disclosure relates to a kit comprising a platinum drug thatis oxaliplatin; a selective OCT2 inhibitor; and instructions for use. Insome embodiments, the present disclosure relates to a kit comprising aplatinum drug that is oxaliplatin; a selective OCT2 inhibitor selectedfrom the group consisting of buflomedil, a buflomedil salt,dolutegravir, and a dolutegravir salt; and instructions for use. The kitmay further comprise instructions for use, e.g., for reducing platinumdrug-induced neurotoxicity; treating cancer; or creasing patientcompliance for treating cancer. The instructions for use are generallywritten instructions, although electronic storage media (e.g., magneticdiskette or optical disk) containing instructions are also acceptable.

The present disclosure relates to a kit comprising a platinum drug thatis cisplatin; a selective OCT2 inhibitor; and instructions for use. Insome embodiments, the present disclosure relates to a kit comprising aplatinum drug that is cisplatin, a selective OCT2 inhibitor selectedfrom the group consisting of buflomedil, a buflomedil salt,dolutegravir, and a dolutegravir salt; and instructions for use. The kitmay further comprise instructions for use, e.g., for reducing platinumdrug-induced neurotoxicity; treating cancer; or creasing patientcompliance for treating cancer. The instructions for use are generallywritten instructions, although electronic storage media (e.g., magneticdiskette or optical disk) containing instructions are also acceptable.

The present disclosure relates to a kit comprising a platinum drug thatis oxaliplatin or cisplatin, a selective OCT2 inhibitor selected fromthe group consisting of buflomedil and a buflomedil salt; andinstructions for use. The kit may further comprise instructions for use,e.g., for reducing platinum drug-induced neurotoxicity; treating cancer;or creasing patient compliance for treating cancer. The instructions foruse are generally written instructions, although electronic storagemedia (e.g., magnetic diskette or optical disk) containing instructionsare also acceptable.

In some embodiments, the kit further comprises a therapeuticallyeffective amount of one or more additional cancer chemotherapeuticagents in addition to the platinum drug. In some embodiments, the one ormore additional chemotherapeutic agents is selected from the groupconsisting of 5-fluorouracil, bevacizumab, capecetabine, gemcitabine,irinotecan, and leucovorin.

The present disclosure also relates to a pharmaceutical kit comprisingone or more containers comprising a platinum drug that is oxaliplatin orcisplatin; and one or more containers comprising a selective OCT2inhibitor selected from the group consisting of buflomedil, a buflomedilsalt, dolutegravir, and a dolutegravir salt. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice reflects approval by the agency for themanufacture, use or sale for human administration. Each component (ifthere is more than one component) can be packaged in separate containersor some components can be combined in one container wherecross-reactivity and shelf life permit. The kits may be in unit dosageforms, bulk packages (e.g., multi-dose packages) or sub-unit doses. Kitsmay also include multiple unit doses of the compounds and instructionsfor use and be packaged in quantities sufficient for storage and use inpharmacies (e.g., hospital pharmacies and compounding pharmacies). Incertain embodiments, multiple dosage forms are provided and formulatedfor different routes of administration.

Also disclosed are articles of manufacture comprising a unit dosage ofthe compositions, in suitable packaging for use in the methods describedherein. Suitable packaging is known in the art and includes, forexample, vials, vessels, ampules, bottles, jars, flexible packaging andthe like. An article of manufacture may further be sterilized and/orsealed. In certain embodiments, multiple compositions are provided andformulated for different routes of administration.

Where the administration involves more than one active agent for use incombination, generally, the agents may be formulated separately or in asingle dosage form, depending on the prescribed most suitableadministration regime for each of the agents concerned.

The instructions can include instructions for determining a desirabledose of the selective OCT2 inhibitor for a subject in need. For example,the instructions can include determining a dose based on at least onefactor of the subject, including, but not limited to, body weight, bodysurface area, height, age, gender, alcohol use, tobacco use, life style,renal function, liver function, genetic polymorphism and co-medications.In some embodiments, the instructions include determining a dose bymonitoring plasma level of the OCT2 inhibitor in the said subject.

Administration of Platinum Drug and OCT2

In some embodiments, the method comprises a step prior to theadministration of the drugs, wherein the step is determining aneffective dose to achieve a plasma level of a selective Organic CationTransporter 2 (OCT2) inhibitor that is buflomedil or a buflomedil saltin the subject in need thereof, wherein the plasma level is two to fivetimes IC₅₀ of the selective Organic Cation Transporter 2 (OCT2)inhibitor that is buflomedil or a buflomedil salt.

In some embodiments, the step of determining an effective dose toachieve a plasma level of a selective Organic Cation Transporter 2(OCT2) inhibitor is performed by administering a pre-dose of theselective Organic Cation Transporter 2 (OCT2) inhibitor. A pre-dose is anormal or low dose. The plasma level can thus be determined for theparticular subject who is dosed.

In some embodiments, the step of determining an effective dose toachieve a plasma level of a selective Organic Cation Transporter 2(OCT2) inhibitor is determined by reference data chart based on subjectcharacteristics. Subject characteristics are particular to an individualand can include body weight, kidney function, liver function, and bodysurface area. A reference data chart based on subject characteristicscan be generated by guidelines from the FDA [FDA, Guidance for Industry:Population Pharmacokinetics, 1999].

In some embodiments, the platinum drug and the selective OCT2 inhibitorare administered at the same time. In some embodiments, the selectiveOCT2 inhibitor is administered before the platinum drug. In someembodiments, the selective OCT2 inhibitor is administered after theplatinum drug.

In some embodiments, the selective OCT2 inhibitor is administered viamore than one route of administration. In some embodiments, the platinumdrug and the selective OCT2 inhibitor are administered via the sameroute of administration.

In some embodiments, the selective OCT2 inhibitor is administered viaintravenous infusion. In some embodiments, the selective OCT2 inhibitoris administered via intravenous injection and intravenous infusion. Insome embodiments, the intravenous infusion is over a period of time atleast 1 hour, such as at least 1, 2, 3, 4, 5 or 6 hours. In someembodiments, the rate of intravenous infusion is constant. In someembodiments, the rate of intravenous infusion is variable.

In some embodiments, the amount of selective OCT2 inhibitor administeredto the subject in need thereof is at least one to five times the OCT2IC₅₀ of the selective OCT2 inhibitor

In some embodiments, the amount of selective OCT2 inhibitor administeredto the subject in need thereof is 50 mg to 600 mg per day.

In some embodiments, the amount of platinum drug administered to thesubject during one treatment session is greater than what isadministered under standard clinical practices.

In some embodiments, the cumulative amount of platinum drug administeredto the subject in need thereof over the entire course of treatment isgreater than what is administered under standard clinical practices.

In some embodiments, the platinum drug is administered at a greaterfrequency than under standard clinical practices.

Enhancing Patient Compliance

The present disclosure provides a method for enhancing patientcompliance. In some embodiments, administration of a platinum drug to asubject would result in pain. In some embodiments, the therapeuticallyeffective amount of platinum drug administered is known to causeplatinum drug-induced toxicity in subjects. Since the compositions areuseful in reducing pain in a subject, a method using the compositionscan achieve increased patient compliance.

In some embodiments, the present disclosure provides methods forincreasing patient compliance for treating cancer in a subject in needthereof comprising administering a therapeutically effective amount of aplatinum drug that is oxaliplatin to the subject in need thereof; andadministering a selective Organic Cation Transporter 2 (OCT2) inhibitorto the subject in need thereof in a dose effective to reduce platinumdrug-induced neurotoxicity in said subject in need thereof, whereby saidsubject completes treatment with a cumulative dose at least 500 mg/m².In some embodiments, subject completes treatment with a cumulative doseat least 500, 600, 700 or 800 mg/m². In some embodiments, subjectcompletes treatment with a cumulative dose at least 500 to 800 mg/m². Insome embodiments, the selective Organic Cation Transporter 2 (OCT2)inhibitor is not buflomedil. In some embodiments, the method furthercomprises a step of determining whether the cancer expresses at leastone of OCT1 or OCT3. In some embodiments, the cancer expresses at leastone of OCT 1 or OCT3.

In some embodiments, the present disclosure provides methods forincreasing patient compliance for treating cancer in a subject in needthereof comprising administering a therapeutically effective amount of aplatinum drug that is cisplatin to the subject in need thereof; andadministering a selective Organic Cation Transporter 2 (OCT2) inhibitorto the subject in need thereof in a dose effective to reduce platinumdrug-induced toxicity in said subject in need thereof, whereby saidsubject completes treatment with a cumulative dose of at least 100mg/m². In some embodiments, subject completes treatment with acumulative dose of 100 mg/m², 200 mg/m², 300 mg/m², 400 mg/m², 500 mg/m²or 600 mg/m². In some embodiments, subject completes treatment with acumulative dose of at least 100 mg/m². In some embodiments, subjectcompletes treatment with a cumulative dose of at least 300 mg/m². Insome embodiments, subject completes treatment with a cumulative dose ofat least 100 to 300 mg/m². In some embodiments, the selective OrganicCation Transporter 2 (OCT2) inhibitor is not buflomedil. In someembodiments, the method further comprises a step of determining whetherthe cancer expresses at least one of OCT1 or OCT3. In some embodiments,the cancer expresses at least one of OCT 1 or OCT3.

In some embodiments, the present disclosure provides method forincreasing patient compliance for treating cancer in a subject in needthereof comprising determining an effective dose to achieve a plasmalevel of a selective Organic Cation Transporter 2 (OCT2) inhibitor thatis buflomedil or a buflomedil salt in the subject in need thereof,wherein the plasma level is two to five times IC₅₀ of the selectiveOrganic Cation Transporter 2 (OCT2) inhibitor that is buflomedil or abuflomedil salt; administering a therapeutically effective amount of aplatinum drug selected from the group consisting of oxaliplatin andcisplatin to the subject in need thereof; and administering a selectiveOrganic Cation Transporter 2 (OCT2) inhibitor that is buflomedil or abuflomedil salt to the subject in need thereof in a dose effective toreduce platinum drug-induced toxicity in said subject in need thereof,whereby said subject completes treatment with a cumulative dose of atleast 100 mg/m². In some embodiments, subject completes treatment with acumulative dose at least 200, 300, 400, 500, 600, 700, or 800 mg/m². Insome embodiments, subject completes treatment with a cumulative dose atleast 500 to 800 mg/m². In some embodiments, subject completes treatmentwith a cumulative dose of at least 100 mg/m². In some embodiments,subject completes treatment with a cumulative dose of at least 300mg/m². In some embodiments, subject completes treatment with acumulative dose of at least 100 to 300 mg/m². In some embodiments, themethod further comprises a step of determining whether the cancerexpresses at least one of OCT1 or OCT3. In some embodiments, the cancerexpresses at least one of OCT 1 or OCT3.

Interim Assessments of Protection Against CIPN to Avoid UnnecessaryContinued Exposure to a Selective OCT2 Inhibitor

In the prevention of CIPN and neuronal cell damage or death due toplatinum drug (e.g., oxaliplatin and cisplatin), it is important tolimit the maximal exposure of a protective agent such as buflomedil anddolutegravir that may have inherent toxicities. These toxicities aremost evident when the plasma level of a selective OCT2 inhibitor exceedsthe safe level defined as C_(H) in FIG. 33, especially if it is exceededrepeatedly. To limit these toxicities it is important to either monitorplasma level of a selective OCT2 inhibitor to ensure it does not exceedC_(H). or to discontinue treatment with the inhibitor if it is found notto be effective on reducing platinum drug (e.g., oxaliplatin orcisplatin) toxicities at some interim point. Since patients require manycycles (typically, 10-12 for oxaliplatin, 4-8 for cisplatin) oftreatment over months, avoiding unnecessary exposure to high plasmalevel of a selective OCT2 inhibitor (e.g., buflomedil and dolutegravir)is important.

Fortunately there are recognized and well validated intermediateobjective measures that can be employed to estimate whether thepreventative therapy reduces peripheral neuropathy caused by oxaliplatinand cisplatin, using methods described in Velasco (Velasco R, Bruna J,Briani C, et al. J Neurol Neurosurg Psychiatry 2014; 85:392-398.). Theinterim point to make these assessments is after as few as 3 or 4 cyclesof platinum drug (e.g., oxaliplatin) treatment. If there is noprotection by these measures then it is futile to continue with aselective OCT2 inhibitor such as buflomedil or dolutegravir. The methodwould employ such methods are described in Velasco 2014. Velasco foundthat three variables obtained at intermediate follow-up, namely, thenumber of acute symptoms (OR 1.9; CI 95% 1.2 to 3.2; p=0.012) andthe >30% decrease in sensory nerve action potential amplitude from thebaseline value in radial (OR 41.4; CI 95% 4.98 to 343.1; p=0.001) anddorsal sural nerves (OR 24.96; CI 95% 2.6 to 239.4; p=0.005) wereindependently associated with the risk of developing severe OXA-IPN. Soif there has been significant deterioration in these parameters after 3,4 or 6 platinum drug (e.g., oxaliplatin) cycles (despite being treatedwith a selective OCT2 inhibitor), discontinuation of administration ofthe selective OCT2 inhibitor (e.g., buflomedil or dolutegravir) at theinterim point to avoid further exposure to an unnecessary andpotentially harmful agent. Similar approach can be applied to cisplatin,which typically triggers peripheral neuropathy after the administrationof 250-350 mg/m² (Argyriou, et. al., Critical Reviews inOncology/Hematology 82(2012)51-77).

The present disclosure provides the disclosed methods, in which theplatinum drug-induced toxicity is assessed after administration of theOCT2 inhibitor. In some embodiments, the present disclosure provides thedisclosed methods, in which the platinum drug-induced toxicity isassessed by establishing the subject's baseline of sensory nerve actionpotential or sensory nerve conduction prior to administration of theplatinum drug. In some embodiments, the platinum drug-induced toxicityis assessed near the midpoint of treatment. In some embodiments, theplatinum drug-induced toxicity is assessed after treatment with acumulative dose of 200 mg/m². In some embodiments, the platinumdrug-induced toxicity is assessed after treatment with a cumulative doseof 200 mg/m², 300 mg/m², 400 mg/m², 500 mg/m², 600 mg/m², 700 mg/m² or800 mg/m².

Medical Uses

As described herein, selective OCT2 inhibitors may be used according tothe teachings herein for use in the treatment of cancer in a subject inneed thereof, wherein the OCT2 inhibitor is for use in combination witha platinum drug selected from the group consisting of oxaliplatin andcisplatin, and with respect to the variations as described for themethods taught herein.

Use of a selective OCT2 inhibitor in the preparation of a medicament forthe treatment of cancer, wherein the medicament is for use incombination with a platinum drug selected from the group consisting ofoxaliplatin and cisplatin is also contemplated based on the teachingsprovided herein and with respect to the variations as described for themethods taught herein.

Use of a platinum drug selected from the group consisting of oxaliplatinand cisplatin in the preparation of a medicament for the treatment ofcancer, wherein the medicament is for use in combination with aselective OCT2 inhibitor is also contemplated based on the teachingsprovided herein and with respect to the variations as described for themethods taught herein.

In certain embodiments of the uses described above, the OCT2 inhibitoris for administration at a dose capable of reducing platinumdrug-induced toxicity. In certain embodiments, the OCT2 inhibitor is foradministration at a dose of between X and Y mg/kg per day.

EXEMPLARY EMBODIMENTS Embodiment I-1

In one embodiment, the present disclosure provides methods for reducingplatinum drug-induced toxicity in a cell expressing Organic CationTransporter 2 (OCT2) comprising

providing a platinum drug to the cell expressing OCT2; andproviding an OCT2 inhibitor that reduces OCT2-mediated platinum druguptake into the cell,

wherein the OCT2 inhibitor is buflomedil or a buflomedil salt andreduces platinum drug induced toxicity in the cell.

Embodiment I-2

In one embodiment, the present disclosure provides a method for reducingplatinum drug-induced toxicity in a cell expressing Organic CationTransporter 2 (OCT2) comprising

providing a platinum drug to the cell expressing OCT2; and

providing an OCT2 inhibitor that reduces OCT2-mediated platinum druguptake into the cell,

-   -   wherein the OCT2 inhibitor is dolutegravir or a dolutegravir        salt and reduces platinum drug-induced toxicity in the cell.

Embodiment I-3

In one embodiment, the present disclosure provides a method for reducingplatinum drug-induced toxicity in a cell expressing Organic CationTransporter 2 (OCT2) comprising

providing a platinum drug to the cell expressing OCT2; andproviding an OCT2 inhibitor that reduces OCT2-mediated platinum druguptake into the cell,wherein the OCT2 inhibitor comprises imidazole and reduces platinumdrug-induced toxicity in the cell.

Embodiment I-4

In a further embodiment of embodiment I-3, the OCT2 inhibitor ismiconazole or a salt thereof.

Embodiment I-5

In a further embodiment of any of embodiments I-1 to I-4, the platinumdrug and the OCT2 inhibitor are provided at the same time.

Embodiment I-6

In a further embodiment of any of embodiments I-1 to I-4, the OCT2inhibitor is provided before the platinum drug.

Embodiment I-7

In a further embodiment of any of embodiments I-1 to I-4, the OCT2inhibitor is provided after the platinum drug.

Embodiment I-8

In a further embodiment of any of embodiments I-1 to I-7, the cellexpressing OCT2 is a cell selected from the group consisting of kidneycell, neuron cell, ear cell, and blood cell.

Embodiment I-9

In a further embodiment of embodiment I-8, the cell is a sensory neuroncell

Embodiment I-10

In a further embodiment of embodiment I-8, wherein the cell is a kidneycell.

Embodiment I-11

In a further embodiment of any of embodiments I-1 to I-10, the platinumdrug is oxaliplatin.

Embodiment I-12

In a further embodiment of any of embodiments I-1 to I-10, the platinumdrug is cisplatin.

Embodiment I-13

In a further embodiment of any of embodiments I-1 to I-10, the platinumdrug is tetraplatin.

Embodiment I-14

In a further embodiment of any of embodiments I-1 to I-13, OCT2-mediatedplatinum drug uptake into the cell expressing OCT2 is inhibited by atleast 50 percent, at least 70 percent or at least 90 percent as comparedto OCT2-mediated platinum drug uptake into a cell not in the presence ofthe OCT2 inhibitor.

Embodiment I-15

In one embodiment, the present disclosure provides a method for reducingplatinum drug-induced toxicity in a subject comprising

providing a platinum drug to a subject comprising cells expressingOrganic Cation Transporter 2 (OCT2); andproviding an OCT2 inhibitor to the subject,wherein the OCT2 inhibitor is buflomedil or a buflomedil salt andreduces OCT2-mediated platinum drug uptake into the cells expressingOCT2, thereby reducing platinum drug-induced toxicity in the subject.

Embodiment I-16

In one embodiment, the present disclosure provides a method for reducingplatinum drug induced toxicity in a subject comprising

providing a platinum drug to a subject comprising cells expressingOrganic Cation Transporter 2 (OCT2); andproviding an OCT2 inhibitor to the subject,wherein the OCT2 inhibitor is dolutegravir or a dolutegravir salt andreduces OCT2 mediated platinum drug uptake into the cells expressingOCT2, thereby reducing platinum drug induced toxicity in the subject.

Embodiment I-17

In one embodiment, the present disclosure provides a method for reducingplatinum drug-induced toxicity in a subject comprising

providing a platinum drug to a subject comprising cells expressingOrganic Cation Transporter 2 (OCT2); andproviding an OCT2 inhibitor to the subject,wherein the OCT2 inhibitor comprises imidazole and reduces OCT2-mediatedplatinum drug uptake into the cells expressing OCT2, thereby reducingplatinum drug-induced toxicity in the subject.

Embodiment I-18

In a further embodiment of embodiment I-17, the OCT2 inhibitor ismiconazole or a salt thereof.

Embodiment I-19

In one embodiment, the present disclosure provides methods for reducingplatinum drug-induced toxicity in a subject, comprising

providing an OCT2 inhibitor to a subject comprising cells expressingOCT2;wherein the OCT2 inhibitor is buflomedil or a buflomedil salt, thesubject has been or will be provided with platinum drug and the OCT2inhibitor reduces OCT2-mediated platinum drug uptake into the cellsexpressing OCT2, thereby reducing platinum drug-induced toxicity in thesubject.

Embodiment I-20

In one embodiment, the present disclosure provides a method for reducingplatinum drug induced toxicity in a subject, comprising

providing an OCT2 inhibitor to a subject comprising cells expressingOCT2;wherein the OCT2 inhibitor is dolutegravir or a dolutegravir salt, thesubject has been or will be provided with platinum drug and the OCT2inhibitor reduces OCT2 mediated platinum drug uptake into the cellsexpressing OCT2, thereby reducing platinum drug induced toxicity in thesubject.

Embodiment I-21

In one embodiment, the present disclosure provides a method for reducingplatinum drug-induced toxicity in a subject, comprising

providing an OCT2 inhibitor to a subject comprising cells expressingOCT2;wherein the OCT2 inhibitor comprises imidazole, the subject has been orwill be provided with platinum drug and the OCT2 inhibitor reducesOCT2-mediated platinum drug uptake into the cells expressing OCT2,thereby reducing platinum drug-induced toxicity in the subject.

Embodiment I-22

In a further embodiment of embodiment I-21, the OCT2 inhibitor ismiconazole or a salt thereof.

Embodiment I-23

In a further embodiment of any of embodiments I-15 to I-22, the platinumdrug and the OCT2 inhibitor are provided at the same time.

Embodiment I-24

In a further embodiment of any of embodiments I-15 to I-22, the OCT2inhibitor is provided before the platinum drug.

Embodiment I-25

In a further embodiment of any of embodiments I-15 to I-22, the OCT2inhibitor is provided after the platinum drug.

Embodiment I-26

In a further embodiment of any of embodiments I-15 to I-25, the platinumdrug is oxaliplatin.

Embodiment I-27

In a further embodiment of any of embodiments I-15 to I-25, the platinumdrug is cisplatin.

Embodiment I-28

In a further embodiment of any of embodiments I-15 to I-25, the platinumdrug is tetraplatin.

Embodiment I-29

In a further embodiment of any of embodiments I-15 to I-28, the amountof OCT2 inhibitor provided to the subject is at a less than atherapeutically effective dosage.

Embodiment I-30

In a further embodiment of any of embodiments I-15 to I-29, the amountof OCT2 inhibitor provided to the subject is 10 mg to 2000 mg per day.

Embodiment I-31

In a further embodiment of any of embodiments I-15 to I-30, the amountof platinum drug provided to the subject during one treatment session isgreater than what is provided under standard clinical practices.

Embodiment I-32

In a further embodiment of any of embodiments I-15 to I-31, thecumulative amount of platinum drug provided to the subject over theentire course of treatment is greater than what is provided understandard clinical practices.

Embodiment I-33

In a further embodiment of any of embodiments I-15 to I-32, the platinumdrug is provided at a greater frequency than under standard clinicalpractices.

Embodiment I-34

In a further embodiment of any of embodiments I-15 to I-33, the platinumdrug-induced toxicity is selected from the group consisting ofnephrotoxicity, neurotoxicity, hematoxicity, and ototoxicity.

Embodiment I-35

In a further embodiment of embodiment I-34, the platinum drug-inducedtoxicity is neurotoxicity.

Embodiment I-36

In a further embodiment of embodiment I-35, the neurotoxicity isperipheral neuropathy.

Embodiment I-37

In a further embodiment of embodiment I-36, the peripheral neuropathy isGrade 3 or Grade 4 peripheral neuropathy.

Embodiment I-38

In a further embodiment of embodiment I-34, the platinum drug-inducedtoxicity is nephrotoxicity.

Embodiment I-39

In a further embodiment of any of embodiments I-15 to I-38, the subjectis a human or a non-human animal.

Embodiment I-40

In a further embodiment of any of embodiments I-15 to I-39, the OCT2inhibitor is provided enterally, intravenously, intramuscularly,intraperitoneally, orally, or parenterally.

Embodiment I-41

In a further embodiment of any of embodiments I-15 to I-40, prevalenceof platinum drug-induced toxicity in a group of subjects is reduced byat least 10% as compared to the prevalence of platinum drug-inducedtoxicity in a group of subjects not provided with an OCT2 inhibitor.

Embodiment I-42

In one embodiment, the present disclosure provides methods for treatingcancer in a subject comprising;

providing a therapeutically effective amount of platinum drug to thesubject having cancer, wherein the subject comprises a cancerous cellexpressing platinum drug uptake transporters not inhibited by an OrganicCation Transporter 2 (OCT2) inhibitor; andproviding an OCT2 inhibitor to the subject,

wherein the OCT2 inhibitor is buflomedil or a buflomedil salt,

thereby treating cancer in the subject.

Embodiment I-43

In one embodiment, the present disclosure provides methods for treatingcancer in a subject comprising;

providing a therapeutically effective amount of platinum drug to thesubject having cancer, wherein the subject comprises a cancerous cellexpressing platinum drug uptake transporters not inhibited by an OrganicCation Transporter 2 (OCT2) inhibitor; andproviding an OCT2 inhibitor to the subject,

wherein the OCT2 inhibitor is dolutegravir or a dolutegravir salt,

thereby treating cancer in the subject.

Embodiment I-44

In one embodiment, the present disclosure provides a method for treatingcancer in a subject comprising;

providing a therapeutically effective amount of platinum drug to thesubject having cancer, wherein the subject comprises a cancerous cellexpressing platinum drug uptake transporters not inhibited by an OrganicCation Transporter 2 (OCT2) inhibitor; andproviding an OCT2 inhibitor to the subject,

wherein the OCT2 inhibitor comprises imidazole,

thereby treating cancer in the subject.

Embodiment I-45

In a further embodiment of embodiment I-44, the OCT2 inhibitor ismiconazole or a salt thereof.

Embodiment I-46

In one embodiment, the present disclosure provides a method forincreasing efficacy of platinum drug treatment in a subject comprising

providing a therapeutically effective amount of platinum drug to thesubject having cancer, wherein the subject comprises a cancerous cellexpressing platinum drug uptake transporters not inhibited by an OCT2inhibitor; andproviding an OCT2 inhibitor to the subject,

wherein the OCT2 inhibitor is buflomedil or a buflomedil salt,

thereby increasing efficacy of the platinum drug treatment.

Embodiment I-47

In one embodiment, the present disclosure provides a method forincreasing efficacy of platinum drug treatment in a subject comprising

providing a therapeutically effective amount of platinum drug to thesubject having cancer, wherein the subject comprises a cancerous cellexpressing platinum drug uptake transporters not inhibited by an OCT2inhibitor; andproviding an OCT2 inhibitor to the subject,

wherein the OCT2 inhibitor is dolutegravir or a dolutegravir salt,

thereby increasing efficacy of the platinum drug treatment.

Embodiment I-48

In one embodiment, the present disclosure provides a method forincreasing efficacy of platinum drug treatment in a subject comprising

providing a therapeutically effective amount of platinum drug to thesubject having cancer, wherein the subject comprises a cancerous cellexpressing platinum drug uptake transporters not inhibited by an OCT2inhibitor; andproviding an OCT2 inhibitor to the subject,

wherein the OCT2 inhibitor comprises imidazole,

thereby increasing efficacy of the platinum drug treatment.

Embodiment I-49

In a further embodiment of embodiment I-48, the OCT2 inhibitor ismiconazole or a salt thereof.

Embodiment I-50

In a further embodiment of any of embodiments I-42 to I-49, the platinumdrug and the OCT2 inhibitor are provided at the same time.

Embodiment I-51

In a further embodiment of any of embodiments I-42 to I-49, the OCT2inhibitor is provided before the platinum drug.

Embodiment I-52

In a further embodiment of any of embodiments I-42 to I-49, the OCT2inhibitor is provided after the platinum drug.

Embodiment I-53

In a further embodiment of any of embodiments I-42 to I-52, the platinumdrug is oxaliplatin.

Embodiment I-54

In a further embodiment of any of embodiments I-42 to I-52, the platinumdrug is cisplatin.

Embodiment I-55

In a further embodiment of any of embodiments I-42 to I-52, the platinumdrug is tetraplatin.

Embodiment I-56

In a further embodiment of any of embodiments I-42 to I-54, the amountof OCT2 inhibitor provided to the subject is at a less than atherapeutically effective dosage.

Embodiment I-57

In a further embodiment of any of embodiments I-42 to I-56, the amountof OCT2 inhibitor provided to the subject is 10 mg to 2000 mg per day.

Embodiment I-58

In a further embodiment of any of embodiments I-42 to I-57, the amountof platinum drug provided to the subject during one treatment session isgreater than what is provided under standard clinical practices.

Embodiment I-59

In a further embodiment of any of embodiments I-42 to I-58, thecumulative amount of platinum drug provided to the subject over theentire course of treatment is greater than what is provided understandard clinical practices.

Embodiment I-60

In a further embodiment of any of embodiments I-42 to I-59, the platinumdrug is provided at a greater frequency than under standard clinicalpractices.

Embodiment I-61

In a further embodiment of any of embodiments I-42 to I-60, the canceris selected from the group consisting of adenocarcinoma of the pancreas,ampullary and periampullary carcinoma, adenocarcinoma of the anus,appendiceal carcinoma, hepatocellular carcinoma, carcinoma of the colonor rectum, epithelial ovarian carcinoma, fallopian tube carcinoma.primary peritoneal cancer, esophageal or esophagogastric junctioncarcinoma, gastric carcinoma, small bowel carcinoma, testicular cancer,cholangiocarcinoma, pancreatic adenocarcinoma, carcinoma of unknownprimary origin, chronic lymphocytic leukemia/small lymphocytic lymphoma,non-Hodgkin's lymphoma, adult T-cell leukemia/lymphoma, AIDS-relatedB-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma,gastric MALT lymphoma, nongastric MALT lymphoma, mantle cell lymphoma,mycosis fungoides/Sezary syndrome, splenic marginal zone lymphoma,peripheral T cell lymphoma, primary cutaneous B-cell lymphoma, primarycutaneous anaplastic large cell lymphoma (ALCL), lung cancer, livercancer, and gallbladder cancer.

Embodiment I-62

In a further embodiment of embodiment I-61, the cancer is carcinoma ofthe colon or rectum.

Embodiment I-63

In a further embodiment of embodiment I-61, the cancer is liver cancer.

Embodiment I-64

In a further embodiment of embodiment I-61, the cancer is lung cancer.

Embodiment I-65

In a further embodiment of any of embodiments I-42 to I-64, the platinumdrug uptake transporters not inhibited by an OCT2 inhibitor are OrganicCation Transporter 1 (OCT1) uptake transporters.

Embodiment I-66

In a further embodiment of any of embodiments I-42 to I-64, the platinumdrug uptake transporters not inhibited by an OCT2 inhibitor are OrganicCation Transporter 3 (OCT3) uptake transporters.

Embodiment I-67

In a further embodiment of any of embodiments I-42 to I-64, the platinumdrug uptake transporters not inhibited by an OCT2 inhibitor are CopperTransporter I (CTR1) uptake transporters.

Embodiment I-68

In a further embodiment of any of embodiments I-42 to I-67, the methodfurther comprises the step of determining whether the cancerous cellexpresses platinum drug uptake transporters not inhibited by an OCT2inhibitor.

Embodiment I-69

In a further embodiment of any of embodiments I-42 to I-68, furthercomprising providing a therapeutically effective amount of one or moreadditional chemotherapeutic agents to the subject in addition to theplatinum drug.

Embodiment I-70

In a further embodiment of embodiment I-69, the one or more additionalchemotherapeutic agents is selected from the group consisting of5-fluorouracil, bevacizumab, capecetabine, gemcitabine, irinotecan, andleucovorin.

Embodiment I-71

In a further embodiment of any of embodiments I-42 to I-70, the subjectis a human or a non-human animal.

Embodiment I-72

In a further embodiment of any of embodiments I-42 to I-71, the OCT2inhibitor is provided enterally, intravenously, intramuscularly,intraperitoneally, orally, or parenterally.

Embodiment I-73

In one embodiment, the present disclosure provides pharmaceuticalcompositions comprising;

a platinum drug,an OCT2 inhibitor, wherein the OCT2 inhibitor is buflomedil or abuflomedil salt; anda pharmaceutically acceptable carrier.

Embodiment I-74

In one embodiment, the present disclosure provides a pharmaceuticalcomposition comprising;

a platinum drug,an OCT2 inhibitor, wherein the OCT2 inhibitor is dolutegravir or adolutegravir salt; anda pharmaceutically acceptable carrier.

Embodiment I-75

In one embodiment, the present disclosure provides a pharmaceuticalcomposition comprising;

a platinum drug,an OCT2 inhibitor, wherein the OCT2 inhibitor comprises imidazole: anda pharmaceutically acceptable carrier.

Embodiment I-76

In a further embodiment of embodiment I-75, the OCT2 inhibitor ismiconazole or a salt thereof.

Embodiment I-77

In a further embodiment of any of embodiments I-73 to I-76, the amountof platinum drug is greater than what is present in a standardpharmaceutical composition comprising platinum drug.

Embodiment I-78

In a further embodiment of any of embodiments I-73 to I-77, furthercomprising a therapeutically effective amount of one or more additionalchemotherapeutic agents in addition to the platinum drug.

Embodiment I-79

In a further embodiment of embodiment I-78, the one or more additionalchemotherapeutic agents is selected from the group consisting of5-fluorouracil, bevacizumab, capecetabine, gemcitabine, irinotecan, andleucovorin.

Embodiment I-80

In a further embodiment of any of embodiments I-73 to I-79, the platinumdrug is oxaliplatin.

Embodiment I-81

In a further embodiment of any of embodiments I-73 to I-79, the platinumdrug is cisplatin.

Embodiment I-82

In a further embodiment of any of embodiments I-73 to I-79, the platinumdrug is tetraplatin.

Embodiment I-83

In a further embodiment of any of embodiments I-3, 1-17, 1-21, I-44, orI-48, the OCT2 inhibitor has a C_(max,u)/IC₅₀ or C_(max)/IC₅₀.app of atleast 1.

Embodiment I-84

In a further embodiment of any of embodiments I-3, I-17, I-21, I-44, orI-48, the OCT2 inhibitor has a C_(max,u)/IC₅₀ or C_(max)/IC_(50,app) ofat least 3.

Embodiment I-85

In a further embodiment of any of embodiments I-3, 1-17, 1-21, I-44,I-48, I-83, or I-84, the OCT2 inhibitor is not toxic to the cell at itsclinical concentration.

Embodiment I-86

In a further embodiment of any of embodiments I-3, 1-17, 1-21, I-44,1-48, or I-83 to I-85, the OCT2 inhibitor does not reduce anti-canceractivity of the platinum drug at its clinical concentration by more than20/6 as compared to anti-cancer activity of the platinum drug at itsclinical concentration in the absence of the OCT2 inhibitor.

Embodiment I-87

In a further embodiment of any of embodiments I-3, I-17, I-21, I-44,I-48, or I-83 to I-86, the OCT2 inhibitor does not reduce uptake of theplatinum drug at its clinical concentration into the cell via othertransporters by more than 20% as compared to uptake of the platinum drugat its clinical concentration via other transporters in the absence ofthe OCT2 inhibitor.

Embodiment I-88

In a further embodiment of any of embodiments I-3, I-17, I-21, I-44,I-48, or I-83 to I-87, the OCT2 inhibitor does not reduce the efflux ofthe platinum drug at its clinical concentration by more than 20% ascompared to the efflux of the platinum drug at its clinicalconcentration in the absence of the OCT2 inhibitor.

Embodiment I-89

In a further embodiment of any of embodiments I-3, I-17, I-21, I-44,I-48, or I-83 to I-88, the OCT2 inhibitor has a mean half-life that isgreater than 2 hours.

Embodiment I-90

In a further embodiment of embodiment I-75, the OCT2 inhibitor has aC_(max,u)/IC₅₀ or C_(max)/IC_(50,app) of at least 1.

Embodiment I-91

In a further embodiment of embodiment I-75, the OCT2 inhibitor has aC_(max,u)/IC₅₀ or C_(max)/IC_(50,app) of at least 3.

Embodiment I-92

In a further embodiment of any of embodiments I-75, I-90, or 1-91, theOCT2 inhibitor is not toxic to the cell at its clinical concentration.

Embodiment I-93

In a further embodiment of any of embodiments I-75 or I-90 to I-92, theOCT2 inhibitor does not reduce anti-cancer activity of the platinum drugat its clinical concentration by more than 20/6 as compared toanti-cancer activity of the platinum drug at its clinical concentrationin the absence of the OCT2 inhibitor.

Embodiment I-94

In a further embodiment of any of embodiments I-75 or I-90 to I-93, theOCT2 inhibitor does not reduce uptake of the platinum drug at itsclinical concentration into the cell via other transporters by more than20% as compared to uptake of the platinum drug at its clinicalconcentration via other transporters in the absence of the OCT2inhibitor.

Embodiment I-95

In a further embodiment of any of embodiments I-75 or I-90 to I-94, theOCT2 inhibitor does not reduce the efflux of the platinum drug at itsclinical concentration by more than 20% as compared to the efflux of theplatinum drug at its clinical concentration in the absence of the OCT2inhibitor.

Embodiment I-96

In a further embodiment of any of embodiments I-75 or I-90 to I-95, theOCT2 inhibitor has a mean half-life that is greater than 2 hours.

Embodiment I-97

In one embodiment, the present disclosure provides a method ofpredicting the efficacy of a platinum drug and Organic CationTransporter 2 (OCT2) inhibitor therapy in a subject having cancercomprising

obtaining a sample comprising at least one cancerous cell; anddetermining whether the cancerous cell expresses platinum drug uptaketransporters not inhibited by an OCT2 inhibitor,wherein platinum drug and OCT2 inhibitor therapy is likely to beeffective in a subject where the cancerous cell expresses platinum druguptake transporters not inhibited by an OCT2 inhibitor.

Embodiment I-98

In one embodiment, the present disclosure provides methods of predictingthe efficacy of platinum drug and Organic Cation Transporter 2 (OCT2)inhibitor therapy in a subject having cancer comprising

obtaining a sample comprising at least one cancerous cell; anddetermining whether the cancerous cell expresses OCT2,

-   -   wherein the platinum drug and OCT2 inhibitor therapy is not        likely to be effective in a subject where the cancerous cell        primarily expresses OCT2.

Embodiment I-99

In a further embodiment of any of embodiments I-97 or I-98, the platinumdrug is oxaliplatin.

Embodiment I-100

In a further embodiment of any of embodiments I-97 or I-98, the platinumdrug is cisplatin.

Embodiment I-101

In a further embodiment of any of embodiments I-97 or I-98, the platinumdrug is tetraplatin.

Embodiment I-102

In a further embodiment of any of embodiments I-97 or I-98, the OCT2inhibitor therapy comprises administering buflomedil or a buflomedilsalt.

Embodiment I-103

In a further embodiment of any of embodiments I-97 or I-98, the OCT2inhibitor therapy comprises administering dolutegravir or a dolutegravirsalt.

Embodiment I-104

In a further embodiment of any of embodiments I-97 or I-98, the OCT2inhibitor therapy comprises administering an OCT2 inhibitor comprisingimidazole.

Embodiment I-105

In a further embodiment of embodiment I-104, the OCT2 inhibitor therapycomprises administering an OCT2 inhibitor comprising miconazole or asalt thereof.

Exemplary Embodiments of Oxaliplatin and Neurotoxicity Embodiment II-1

In one embodiment, the present disclosure provides methods for reducingplatinum drug-induced neurotoxicity in a subject in need thereofcomprising

administering a platinum drug that is oxaliplatin to the subject in needthereof; and

administering an effective dose of a selective Organic CationTransporter 2 (OCT2) inhibitor to the subject in need thereof,

wherein the subject in need thereof has a cancer.

Embodiment II-2

In one embodiment, the present disclosure provides methods for treatingcancer in a subject in need thereof comprising

administering a therapeutically effective amount of a platinum drug thatis oxaliplatin to the subject in need thereof; and

administering an effective dose of a selective Organic CationTransporter 2 (OCT2) inhibitor to the subject in need thereof.

Embodiment II-3

In one embodiment, the present disclosure provides methods forincreasing patient compliance for treating cancer in a subject in needthereof comprising

administering a therapeutically effective amount of a platinum drug thatis oxaliplatin to the subject in need thereof; and

administering a selective Organic Cation Transporter 2 (OCT2) inhibitorto the subject in need thereof in a dose effective to reduce platinumdrug-induced neurotoxicity in said subject in need thereof, whereby saidsubject completes treatment with a cumulative dose at least 500 mg/m².

Embodiment II-4

The method of Embodiments II-2 or 3, wherein the therapeuticallyeffective amount of platinum drug administered is known to causeplatinum drug-induced neurotoxicity in subjects.

Embodiment II-5

The method of any of Embodiments II-1 or 3-4, wherein the neurotoxicityis peripheral neurotoxicity.

Embodiment II-6

The method of any of Embodiments II-1 or 3-4, wherein the neurotoxicityis damage to a sensory neuron.

Embodiment II-7

The method of any of Embodiments II-1 or 3-4, wherein the neurotoxicityis damage to a motor neuron.

Embodiment II-7a

The method of any of Embodiments II-1-7, wherein the neurotoxicity ischronic neurotoxicity.

Embodiment II-7b

The method of any of Embodiments II-1-7, wherein the neurotoxicity isacute syndrome transient neurotoxicity.

Embodiment II-7c

The method of any of Embodiments II-1-7, wherein the neurotoxicityoccurs 1 hour to seven days after first treatment.

Embodiment II-7d

The method of any of Embodiments II-1-7, wherein the neurotoxicityoccurs after a subject completes treatment with a cumulative dose of atleast 500 mg/m²

Embodiment II-8

The method of any of Embodiments II-1 or 3-4, wherein the neurotoxicityis damage to dorsal root ganglia (DRG).

Embodiment II-9

The method of any of Embodiments II-1-8, wherein the dose of selectiveOCT2 inhibitor is effective to minimize platinum drug-inducedneurotoxicity in said subject in need thereof.

Embodiment II-10

The method of any of Embodiments II-1-8, wherein the dose of selectiveOCT2 inhibitor is effective to prevent platinum drug-inducedneurotoxicity of Grade 3 or higher, as assessed by a method selectedfrom National Cancer Institute-Common Toxicity Criteria (NCI-CTC)sensory scale, National Cancer Institute-Common Toxicity Criteria(NCI-CTC) motor scale, Total Neuropathy Score clinical version (TNSc)and European Organization for Research and Treatment of Cancer CIPNspecific self-report questionnaire (EORTC QOL-CIPN20), in patientstreated with a cumulative dose of at least 100 mg/m².

Embodiment II-11

The method of any of Embodiments II-1-8, wherein the dose of selectiveOCT2 inhibitor is effective to prevent platinum drug-inducedneurotoxicity of Grade 3 or higher, as assessed by a method selectedfrom National Cancer Institute-Common Toxicity Criteria (NCI-CTC)sensory scale, National Cancer Institute-Common Toxicity Criteria(NCI-CTC) motor scale, Total Neuropathy Score clinical version (TNSc)and European Organization for Research and Treatment of Cancer CIPNspecific self-report questionnaire (EORTC QOL-CIPN20), in patientstreated with a platinum drug dose intensity of at least 30 mg/week/m².

Embodiment II-12

The method of any of Embodiments II-1-8, wherein the dose of selectiveOCT2 inhibitor is effective to prevent platinum drug-inducedneurotoxicity of Grade 3 or higher, as assessed by a method selectedfrom National Cancer Institute-Common Toxicity Criteria (NCI-CTC)sensory scale, National Cancer Institute-Common Toxicity Criteria(NCI-CTC) motor scale, Total Neuropathy Score clinical version (TNSc)and European Organization for Research and Treatment of Cancer CIPNspecific self-report questionnaire (EORTC QOL-CIPN20), in patientstreated with a platinum drug dose of at least 80 mg/m².

Embodiment II-13

The method of any of Embodiments II-1-12, further comprising assessingplatinum drug-induced neurotoxicity in a subject after administration ofthe platinum drug.

Embodiment II-14

The method of Embodiment II-13, wherein the neurotoxicity is assessed bya method selected from National Cancer Institute-Common ToxicityCriteria (NCI-CTC) sensory scale, National Cancer Institute-CommonToxicity Criteria (NCI-CTC) motor scale, Total Neuropathy Score clinicalversion (TNSc) and European Organization for Research and Treatment ofCancer CIPN specific self-report questionnaire (EORTC QOL-CIPN20).

Embodiment II-15

The method of Embodiment II-13, wherein the neurotoxicity is assessed bya measurement selected from sensory nerve action potential, sensorynerve conduction velocity, cold pain threshold, heat pain threshold,mechanical pain threshold, cold detection threshold, warm detectionthreshold, mechanical detection threshold, vibration perceptionthreshold, current perception threshold, pinprick sensibility, deeptendon reflexes and grip strength.

Embodiment II-16

The method of Embodiment II-13, wherein the neurotoxicity is assessed bymeasuring sensory nerve action potential in one of radial, dorsal sural,sural and ulnar nerves.

Embodiment II-17

The method of any of Embodiments II-13-16, further compromisingestablishing the subject's baseline prior to administration of theplatinum drug.

Embodiment II-18

The method of any of Embodiments II-13-16, wherein the platinumdrug-induced neurotoxicity is assessed near the midpoint of treatment.

Embodiment II-19

The method of any of Embodiments II-13-16, wherein the platinumdrug-induced neurotoxicity is assessed after treatment with a cumulativedose of 200 mg/m²

Embodiment II-20

The method of any of Embodiments II-1-8, wherein the selective OCT2inhibitor is selected from the group consisting of buflomedil, abuflomedil salt, dolutegravir, and a dolutegravir salt.

Embodiment II-21

The method of Embodiment II-20, wherein the selective OCT2 inhibitor isbuflomedil, or a buflomedil salt.

Embodiment II-22

The method of Embodiment II-21, wherein the dose of buflomedil or abuflomedil salt administered in a subject is adjusted based on at leastone of the factors of the said subject, body weight, body surface area,height, age, gender, alcohol use, tobacco use, life style, renalfunction, liver function, genetic polymorphism and co-medications.

Embodiment II-23

The method of Embodiments II-21 or 22, wherein the dose of buflomedil ora buflomedil salt results in its plasma level during the period ofplatinum administration at least 0.43 mg/l, 0.86 mg/l, 1.29 mg/l, 1.72mg/l or 2.15 mg/l.

Embodiment II-24

The method of any of Embodiments II-21-23, wherein the dose ofbuflomedil or a buflomedil salt is at least 100 mg, 200 mg, 300 mg, 400mg, 500 mg or 600 mg.

Embodiment II-25

The method of any of Embodiments II-21-24, wherein the dose ofbuflomedil or a buflomedil salt is at least 1 mg/kg, 2 mg/kg, 3 mg/kg, 4mg/kg, 5 mg/kg, 6 mg/kg, 8 mg/kg or 10 mg/kg.

Embodiment II-26

The method of Embodiment II-20, wherein the selective OCT2 inhibitor isdolutegravir or a dolutegravir salt.

Embodiment II-27

The method of Embodiment II-26, wherein the dose of dolutegravir or adolutegravir salt results in its plasma level during the period ofplatinum administration at least 1.4 mg/l, 2.8 mg/l, 4.2 mg/l, 5.6 mg/lor 7.0 mg/l.

Embodiment II-28

The method of Embodiment II-26 or 27, wherein the dose of dolutegraviror a dolutegravir salt is at least 50 mg, 100 mg, 150 mg, 200 mg, 250mg, 300 mg or 400 mg.

Embodiment II-29

The method of any of Embodiments II-26-28, wherein the dose ofdolutegravir or a dolutegravir salt is at least 1 mg/kg, 2 mg/kg, 3mg/kg, 4 mg/kg, 5 mg/kg or 6 mg/kg.

Embodiment II-30

The method of any of Embodiments II-1-29, wherein the selective OCT2inhibitor does not reduce the efficacy of the platinum drug.

Embodiment II-31

The method of any of Embodiments II-1-30, wherein the selective OCT2inhibitor is administered at a dose that results in its plasmaconcentration during the period of platinum drug administration lessthan its maximum tolerated plasma concentration (MTC) and greater than1×, 2×, 3×, 4× of its IC₅₀ value for OCT2-mediated transport of 20 μMoxaliplatin assessed in human serum or an assay buffer containing 4%bovine serum albumin.

Embodiment II-32

The method of any of Embodiments II-1-19, 30 or 31, wherein theselective OCT2 inhibitor has an IC₅₀ for OCT2-mediated transport of 20μM oxaliplatin in human serum or an assay solution containing 4% bovineserum albumin, of less than or equal to 2 μM.

Embodiment II-33

The method of Embodiment II-32, wherein the selective OCT2 inhibitor hasan IC₅₀ for OCT2-mediated transport of 20 μM oxaliplatin in human serumor an assay solution containing 4% bovine serum albumin that is at least15-fold less than the selective OCT2 inhibitor IC₅₀ for 20 μMoxaliplatin transport in human serum or an assay solution containing 4%bovine serum albumin mediated by OCT1-, OCT-3, and MATE-1.

Embodiment II-34

The method of any of Embodiments II-1-33, wherein the subject in needthereof has a cancer expressing at least one of Organic CationTransporter 1 (OCT1) or Organic Cation Transporter 3 (OCT3).

Embodiment II-35

The method of any of Embodiments II-1-25 or 30-34, wherein the selectiveOCT2 inhibitor is buflomedil, or a buflomedil salt.

Embodiment II-36

The method of any of Embodiments II-1-20 or 26-34, wherein the selectiveOCT2 inhibitor is dolutegravir, or a dolutegravir salt.

Embodiment II-37

The method of any of Embodiments II-1-36, wherein the platinum drug andthe selective OCT2 inhibitor are administered at the same time.

Embodiment II-38

The method of any of Embodiments I-1-36, wherein the selective OCT2inhibitor is administered before the platinum drug.

Embodiment II-39

The method of any of Embodiments II-1-36, wherein the selective OCT2inhibitor is administered after the platinum drug.

Embodiment II-40

The method of any of Embodiments II-1-39, wherein the amount of platinumdrug administered to the subject during one treatment session is greaterthan what is administered under standard clinical practices.

Embodiment II-41

The method of any of Embodiments II-1-40, wherein the cumulative amountof platinum drug administered to the subject in need thereof over theentire course of treatment is greater than what is administered understandard clinical practices.

Embodiment II-42

The method of any of Embodiments II-141, wherein the platinum drug isadministered at a greater frequency than under standard clinicalpractices.

Embodiment II-43

The method of any of Embodiments II-1 or 3-42, wherein the neurotoxicityis peripheral neuropathy.

Embodiment II-44

The method of Embodiment II-43, wherein the peripheral neuropathy isGrade 3 or Grade 4 peripheral neuropathy

Embodiment II-45

The method of any of Embodiments II-1-44, wherein the subject in needthereof is a human or a non-human animal.

Embodiment I-46

The method of any of Embodiments II-1-45, wherein the selective OCT2inhibitor is administered enterally, intravenously, intramuscularly,intraperitoneally, orally, or parenterally.

Embodiment II-47

The method of any of Embodiments II-1-46, wherein the selective OCT2inhibitor is administered via more than one route of administration.

Embodiment II-48

The method of any of Embodiments II-1-46, wherein the platinum drug andthe selective OCT2 inhibitor are administered via the same route ofadministration.

Embodiment II-49

The method of any of Embodiments II-1-45, wherein the selective OCT2inhibitor is administered via intravenous infusion.

Embodiment II-50

The method of any of Embodiments II-1-45, wherein the selective OCT2inhibitor is administrated via intravenous injection and intravenousinfusion.

Embodiment II-51

The method of Embodiments II-49 or 50, wherein intravenous infusion isover a period of time at least 1 hour.

Embodiment II-52

The method of Embodiments II-49 or 50, wherein the rate of intravenousinfusion is constant.

Embodiment II-53

The method of Embodiments II-49 or 50, wherein the rate of intravenousinfusion is variable.

Embodiment II-54

The method of any of Embodiments II-1-53, wherein the cancer expressesOCT1.

Embodiment II-55

The method of any of Embodiments II-1-54, wherein the cancer expressesOCT3.

Embodiment II-56

The method of any of Embodiments II-1-55, wherein the cancer expressesOCT1 and OCT3.

Embodiment II-57

The method of any one of Embodiments II-1-56, wherein the cancer isselected from the group consisting of adenocarcinoma of the pancreas,ampullary and periampullary carcinoma, adenocarcinoma of the anus,appendiceal carcinoma, hepatocellular carcinoma, carcinoma of the colonor rectum, epithelial ovarian carcinoma, fallopian tube carcinoma.primary peritoneal cancer, esophageal or esophagogastric junctioncarcinoma, gastric carcinoma, small bowel carcinoma, testicular cancer,cholangiocarcinoma, pancreatic adenocarcinoma, carcinoma of unknownprimary origin, chronic lymphocytic leukemia/small lymphocytic lymphoma,non-Hodgkin's lymphoma, adult T-cell leukemia/lymphoma, AIDS-relatedB-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma,gastric MALT lymphoma, nongastric MALT lymphoma, mantle cell lymphoma,mycosis fungoides/Sezary syndrome, splenic marginal zone lymphoma,peripheral T cell lymphoma, primary cutaneous B-cell lymphoma, primarycutaneous anaplastic large cell lymphoma (ALCL), lung cancer, livercancer, head and neck cancer, prostate cancer, smooth muscle cancer andgallbladder cancer.

Embodiment II-58

The method of Embodiment II-57, wherein the cancer is ovarian cancer.

Embodiment II-59

The method of Embodiment II-57, wherein the cancer is head and neckcancer.

Embodiment II-60

The method of Embodiment II-57, wherein the cancer is prostate cancer.

Embodiment II-61

The method of Embodiment II-57, wherein the cancer is lymphoma.

Embodiment II-62

The method of Embodiment II-57, wherein the cancer is smooth musclecancer.

Embodiment II-63

The method of Embodiment II-57, wherein the cancer is carcinoma of thecolon or rectum.

Embodiment II-64

The method of Embodiment II-57, wherein the cancer is liver cancer.

Embodiment II-65

The method of Embodiment II-57, wherein the cancer is lung cancer.

Embodiment II-66

The method of any of Embodiments II-1-65, further comprisingadministering to the subject in need thereof a therapeutically effectiveamount of one or more additional cancer chemotherapeutic agents inaddition to the platinum drug.

Embodiment II-67

The method of Embodiment II-66, wherein the one or more additionalchemotherapeutic agents is selected from the group consisting of5-fluorouracil, bevacizumab, capecetabine, gemcitabine, irinotecan, andleucovorin.

Embodiment II-68

A pharmaceutical composition formulated for intravenous administrationcomprising

a platinum drug that is oxaliplatin,

a selective OCT2 inhibitor selected from the group consisting ofbuflomedil, a buflomedil salt, dolutegravir, and a dolutegravir salt,and

a pharmaceutically acceptable carrier.

Embodiment II-69

The pharmaceutical composition of Embodiment II-68, wherein theselective OCT2 inhibitor is buflomedil, or a buflomedil salt.

Embodiment II-70

The pharmaceutical composition of Embodiment II-68, wherein the OCT2inhibitor is dolutegravir, or a dolutegravir salt.

Embodiment II-71

The pharmaceutical composition of any of Embodiments II-68-70, whereinthe amount of the platinum drug is greater than what is present in astandard pharmaceutical composition comprising the platinum drug.

Embodiment II-72

The pharmaceutical composition of any of Embodiments II-68-71, furthercomprising a therapeutically effective amount of one or more additionalcancer chemotherapeutic agents in addition to the platinum drug.

Embodiment II-73

The pharmaceutical composition of Embodiment II-72, wherein the one ormore additional chemotherapeutic agents is selected from the groupconsisting of 5-fluorouracil, bevacizumab, capecetabine, gemcitabine,irinotecan, and leucovorin.

Embodiment II-74

A kit comprising a therapeutically effective amount of a platinum drugthat is oxaliplatin; a selective OCT2 inhibitor and instructions foruse.

Embodiment II-75

The kit of Embodiment II-74, further comprising instructions fordetermining a desirable dose of the selective OCT2 inhibitor for asubject in need.

Embodiment II-76

The kit of Embodiment II-75, wherein the dose is determined based on atleast one factor of the said subject selected from body weight, bodysurface area, height, age, gender, alcohol use, tobacco use, life style,renal function, liver function, genetic polymorphism and co-medications.

Embodiment II-77

The kit of Embodiment II-75, wherein the dose is determined bymonitoring plasma level of the OCT2 inhibitor in the said subject.

Embodiment II-78

The kit of any one of Embodiments II-74-77, wherein the selective OCT2inhibitor is buflomedil or a buflomedil salt.

Embodiment II-79

The kit of Embodiment II-78, wherein buflomedil or a buflomedil salt ispresent in an amount greater than 300 mg, 450 mg, 600 mg, 800 mg or 1000mg.

Embodiment II-80

The kit of any of Embodiments II-74-77, wherein the selective OCT2inhibitor is dolutegravir or a dolutegravir salt.

Embodiment II-81

The kit of Embodiment II-80, wherein dolutegravir or a dolutegravir saltis present in an amount greater than 50 mg, 75 mg, 100 mg, 150 mg, 200mg or 300 mg.

Embodiment II-82

The kit of any of Embodiments II-74-81, wherein the instructions statethat the kit is intended for use in reducing platinum drug-inducedneurotoxicity.

Embodiment II-83

The kit of Embodiment II-74-80, wherein the instructions state that thekit is intended for use in treating cancer.

Embodiment II-84

The kit of any of Embodiments II-74-83, further comprising atherapeutically effective amount of one or more additional cancerchemotherapeutic agents in addition to the platinum drug.

Embodiment II-85

The kit of Embodiment II-84, wherein the one or more additionalchemotherapeutic agents is selected from the group consisting of5-fluorouracil, bevacizumab, capecetabine, gemcitabine, irinotecan, andleucovorin.

Exemplary Embodiments of Cisplatin and Toxicities Embodiment III-1

In one embodiment, the present disclosure provides methods for reducingplatinum drug-induced toxicity in a subject in need thereof comprising

administering a platinum drug that is cisplatin to the subject in needthereof; and

administering an effective dose of a selective Organic CationTransporter 2 (OCT2) inhibitor to the subject in need thereof,

wherein the toxicity is nephrotoxicity, ototoxicity or peripheralneuropathy, and

wherein the subject in need thereof has a cancer.

Embodiment III-2

In one embodiment, the present disclosure provides methods for treatingcancer in a subject in need thereof comprising

administering a therapeutically effective amount of a platinum drug thatis cisplatin to the subject in need thereof; and

administering an effective dose of a selective Organic CationTransporter 2 (OCT2) inhibitor to the subject in need thereof.

Embodiment III-3

In one embodiment, the present disclosure provides methods forincreasing patient compliance for treating cancer in a subject in needthereof comprising

administering a therapeutically effective amount of a platinum drug thatis cisplatin to the subject in need thereof; and

administering a selective Organic Cation Transporter 2 (OCT2) inhibitorto the subject in need thereof in a dose effective to reduce platinumdrug-induced toxicity in said subject in need thereof, whereby saidsubject completes treatment with a cumulative dose of at least 100mg/m².

Embodiment III-4

The method of Embodiment III-2 or 3, wherein the therapeuticallyeffective amount of platinum drug administered is known to causeplatinum drug-induced neurotoxicity in subjects.

Embodiment III-5

The method of Embodiment III-4, wherein the neurotoxicity is peripheralneurotoxicity.

Embodiment III-6

The method of Embodiment II-4, wherein the neurotoxicity is damage to asensory neuron.

Embodiment III-7

The method of Embodiment III-4, wherein the neurotoxicity is damage to amotor neuron.

Embodiment III-7a

The method of any of Embodiments III-1-7, wherein the neurotoxicity ischronic neurotoxicity.

Embodiment III-7b

The method of any of Embodiments III-1-7, wherein the neurotoxicity isacute syndrome transient neurotoxicity.

Embodiment III-7c

The method of any of Embodiments III-1-7, wherein the neurotoxicityoccurs 1 hour to seven days after first treatment.

Embodiment III-7d

The method of any of Embodiments III-1-7, wherein the neurotoxicityoccurs after a subject completes treatment with a cumulative dose of atleast 500 mg/m²

Embodiment III-8

The method of Embodiment III-4, wherein the neurotoxicity is damage todorsal root ganglia (DRG).

Embodiment III-9

The method of any of Embodiments III-1-8, wherein the dose of selectiveOCT2 inhibitor is effective to minimize platinum drug-inducedneurotoxicity in said subject in need thereof.

Embodiment III-10

The method of any of Embodiments III-1-8, wherein the dose of selectiveOCT2 inhibitor is effective to prevent platinum drug-inducedneurotoxicity of Grade 3 or higher, as assessed by a method selectedfrom National Cancer Institute-Common Toxicity Criteria (NCI-CTC)sensory scale, National Cancer Institute-Common Toxicity Criteria(NCI-CTC) motor scale, Total Neuropathy Score clinical version (TNSc)and European Organization for Research and Treatment of Cancer CIPNspecific self-report questionnaire (EORTC QOL-CIPN20), in patientstreated with a cumulative dose of at least 100 mg/m².

Embodiment III-11

The method of any of Embodiments III-1-8, wherein the dose of selectiveOCT2 inhibitor is effective to prevent platinum drug-inducedneurotoxicity of Grade 3 or higher, as assessed by a method selectedfrom National Cancer Institute-Common Toxicity Criteria (NCI-CTC)sensory scale, National Cancer Institute-Common Toxicity Criteria(NCI-CTC) motor scale, Total Neuropathy Score clinical version (TNSc)and European Organization for Research and Treatment of Cancer CIPNspecific self-report questionnaire (EORTC QOL-CIPN20), in patientstreated with a platinum drug dose intensity of at least 30 mg/week/m².

Embodiment III-12

The method of any of Embodiments III-1-8, wherein the dose of selectiveOCT2 inhibitor is effective to prevent platinum drug-inducedneurotoxicity of Grade 3 or higher, as assessed by a method selectedfrom National Cancer Institute-Common Toxicity Criteria (NCI-CTC)sensory scale, National Cancer Institute-Common Toxicity Criteria(NCI-CTC) motor scale, Total Neuropathy Score clinical version (TNSc)and European Organization for Research and Treatment of Cancer CIPNspecific self-report questionnaire (EORTC QOL-CIPN20), in patientstreated with a platinum drug dose of at least 80 mg/m².

Embodiment III-13

The method of any of Embodiments III-1-12, further comprising assessingplatinum drug-induced neurotoxicity in a subject after administration ofthe platinum drug.

Embodiment III-14

The method of Embodiment III-13, wherein the neurotoxicity is assessedby a method selected from National Cancer Institute-Common ToxicityCriteria (NCI-CTC) sensory scale, National Cancer Institute-CommonToxicity Criteria (NCI-CTC) motor scale, Total Neuropathy Score clinicalversion (TNSc) and European Organization for Research and Treatment ofCancer CIPN specific self-report questionnaire (EORTC QOL-CIPN20).

Embodiment III-15

The method of Embodiment III-13, wherein the neurotoxicity is assessedby a measurement selected from sensory nerve action potential, sensorynerve conduction velocity, cold pain threshold, heat pain threshold,mechanical pain threshold, cold detection threshold, warm detectionthreshold, mechanical detection threshold, vibration perceptionthreshold, current perception threshold, pinprick sensibility, deeptendon reflexes and grip strength.

Embodiment III-16

The method of Embodiment III-13, wherein the neurotoxicity is assessedby measuring sensory nerve action potential in one of radial, dorsalsural, sural and ulnar nerves.

Embodiment III-17

The method of any of Embodiments III-13-16, further compromisingestablishing the subject's baseline prior to administration of theplatinum drug.

Embodiment III-18

The method of any of Embodiments III-13-16, wherein the platinumdrug-induced neurotoxicity is assessed near the midpoint of treatment.

Embodiment III-19

The method of any of Embodiments III-13-16, wherein the platinumdrug-induced neurotoxicity is assessed after treatment with a cumulativedose of 200 mg/m².

Embodiment III-20

The method of Embodiment III-1, wherein toxicity is nephrotoxicity.

Embodiment III-21

The method of any of Embodiments III-1-8, wherein the selective OCT2inhibitor is selected from the group consisting of buflomedil, abuflomedil salt, dolutegravir, and a dolutegravir salt.

Embodiment III-22

The method of Embodiment III-21, wherein the selective OCT2 inhibitor isbuflomedil, or a buflomedil salt.

Embodiment III-23

The method of Embodiment III-22, wherein the dose of buflomedil or abuflomedil salt administered in a subject is adjusted based on at leastone of the factors of the said subject, body weight, body surface area,height, age, gender, alcohol use, tobacco use, life style, renalfunction, liver function, genetic polymorphism and co-medications.

Embodiment III-24

The method of Embodiments III-22 or 23, wherein the dose of buflomedilor a buflomedil salt results in its plasma level during the period ofplatinum administration at least 0.43 mg/l, 0.86 mg/l, 1.29 mg/l, 1.72mg/l or 2.15 mg/l.

Embodiment III-25

The method of any of Embodiments III-22-24, wherein the dose ofbuflomedil or a buflomedil salt is at least 100 mg, 200 mg, 300 mg, 400mg, 500 mg or 600 mg.

Embodiment III-26

The method of any of Embodiments III-22-25, wherein the dose ofbuflomedil or a buflomedil salt is at least 1 mg/kg, 2 mg/kg, 3 mg/kg, 4mg/kg, 5 mg/kg, 6 mg/kg, 8 mg/kg or 10 mg/kg.

Embodiment III-27

The method of Embodiment III-21, wherein the selective OCT2 inhibitor isdolutegravir or a dolutegravir salt.

Embodiment III-28

The method of Embodiment III-27, wherein the dose of dolutegravir or adolutegravir salt results in its plasma level during the period ofplatinum administration at least 1.4 mg/l, 2.8 mg/l, 4.2 mg/l, 5.6 mg/lor 7.0 mg/l.

Embodiment III-29

The method of Embodiment III-27 or 28, wherein the dose of dolutegraviror a dolutegravir salt is at least 50 mg, 100 mg, 150 mg, 200 mg, 250mg, 300 mg or 400 mg.

Embodiment III-30

The method of any of Embodiments III-27-29, wherein the dose ofdolutegravir or a dolutegravir salt is at least 1 mg/kg, 2 mg/kg, 3mg/kg, 4 mg/kg, 5 mg/kg or 6 mg/kg.

Embodiment III-31

The method of any of Embodiments III-1-30, wherein the selective OCT2inhibitor does not reduce the efficacy of the platinum drug.

Embodiment III-32

The method of any of Embodiments III-1-31, wherein the selective OCT2inhibitor is administered at a dose that results in its plasmaconcentration during the period of platinum drug administration lessthan its maximum tolerated plasma concentration (MTC) and greater than1×, 2×, 3×, 4× of its IC₅₀ value for OCT2-mediated transport of 20 μMoxaliplatin assessed in human serum or an assay buffer containing 4%bovine serum albumin.

Embodiment III-33

The method of any of Embodiments III-1-19, 31 or 32, wherein theselective OCT2 inhibitor has an IC₅₀ for OCT2-mediated transport of 20μM oxaliplatin in human serum or an assay solution containing 4% bovineserum albumin, of less than or equal to 5 μM.

Embodiment III-34

The method of Embodiment III-33, wherein the selective OCT2 inhibitorhas an IC₅₀ for OCT2-mediated transport of 20 μM oxaliplatin in humanserum or an assay solution containing 4% bovine serum albumin that is atleast 10-fold less than the selective OCT2 inhibitor IC₅₀ for 20 μMoxaliplatin transport in human serum or an assay solution containing 4%bovine serum albumin mediated by one or more of OCT1-, OCT-3, or MATE-1.

Embodiment III-35

The method of any of Embodiments III-1-34, wherein the subject in needthereof has a cancer expressing at least one of Organic CationTransporter 1 (OCT1) or Organic Cation Transporter 3 (OCT3).

Embodiment III-36

The method of any of Embodiments III-1-26 or 31-35, wherein theselective OCT2 inhibitor is buflomedil, or a buflomedil salt.

Embodiment III-37

The method of any of Embodiments III-1-20 or 27-35, wherein theselective OCT2 inhibitor is dolutegravir, or a dolutegravir salt.

Embodiment III-38

The method of any of Embodiments III-1-37, wherein the platinum drug andthe selective OCT2 inhibitor are administered at the same time.

Embodiment III-39

The method of any of Embodiments III-1-37, wherein the selective OCT2inhibitor is administered before the platinum drug.

Embodiment III-40

The method of any of Embodiments III-1-37, wherein the selective OCT2inhibitor is administered after the platinum drug.

Embodiment III-41

The method of any of Embodiments III-1-40, wherein the amount ofplatinum drug administered to the subject during one treatment sessionis greater than what is administered under standard clinical practices.

Embodiment III-42

The method of any of Embodiments III-1-41, wherein the cumulative amountof platinum drug administered to the subject in need thereof over theentire course of treatment is greater than what is administered understandard clinical practices.

Embodiment III-43

The method of any of Embodiments III-1-42, wherein the platinum drug isadministered at a greater frequency than under standard clinicalpractices.

Embodiment III-44

The method of any of Embodiments III-1 or 3-43, wherein theneurotoxicity is peripheral neuropathy.

Embodiment III-45

The method of Embodiment III-44, wherein the peripheral neuropathy isGrade 3 or Grade 4 peripheral neuropathy.

Embodiment III-46

The method of any of Embodiments III-145, wherein the subject in needthereof is a human or a non-human animal.

Embodiment III-47

The method of any of Embodiments III-146, wherein the selective OCT2inhibitor is administered enterally, intravenously, intramuscularly,intraperitoneally, orally, or parenterally.

Embodiment III-48

The method of any of Embodiments III-1-47, wherein the selective OCT2inhibitor is administered via more than one route of administration.

Embodiment III-49

The method of any of Embodiments III-147, wherein the platinum drug andthe selective OCT2 inhibitor are administered via the same route ofadministration.

Embodiment III-50

The method of any of Embodiments III-147, wherein the selective OCT2inhibitor is administered via intravenous infusion.

Embodiment III-51

The method of any of Embodiments III-147, wherein the selective OCT2inhibitor is administrated via intravenous injection and intravenousinfusion.

Embodiment III-52

The method of Embodiments III-50 or 51, wherein intravenous infusion isover a period of time at least 1 hour.

Embodiment III-53

The method of Embodiments III-50 or 51, wherein the rate of intravenousinfusion is constant.

Embodiment III-54

The method of Embodiments III-50 or 51, wherein the rate of intravenousinfusion is variable.

Embodiment III-55

The method of any of Embodiments III-1-54, wherein the cancer expressesOCT 1.

Embodiment III-56

The method of any of Embodiments III-1-55, wherein the cancer expressesOCT3.

Embodiment III-57

The method of any of Embodiments III-1-56, wherein the cancer expressesOCT1 and OCT3.

Embodiment III-58

The method of any one of Embodiments III-1-57, wherein the cancer isselected from the group consisting of adenocarcinoma of the pancreas,ampullary and periampullary carcinoma, adenocarcinoma of the anus,appendiceal carcinoma, hepatocellular carcinoma, carcinoma of the colonor rectum, epithelial ovarian carcinoma, fallopian tube carcinoma.primary peritoneal cancer, esophageal or esophagogastric junctioncarcinoma, gastric carcinoma, small bowel carcinoma, testicular cancer,cholangiocarcinoma, pancreatic adenocarcinoma, carcinoma of unknownprimary origin, chronic lymphocytic leukemia/small lymphocytic lymphoma,non-Hodgkin's lymphoma, adult T-cell leukemia/lymphoma, AIDS-relatedB-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma,gastric MALT lymphoma, nongastric MALT lymphoma, mantle cell lymphoma,mycosis fungoides/Sezary syndrome, splenic marginal zone lymphoma,peripheral T cell lymphoma, primary cutaneous B-cell lymphoma, primarycutaneous anaplastic large cell lymphoma (ALCL), lung cancer, livercancer, head and neck cancer, prostate cancer, smooth muscle cancer andgallbladder cancer.

Embodiment III-59

The method of Embodiment III-58, wherein the cancer is ovarian cancer.

Embodiment III-60

The method of Embodiment III-58, wherein the cancer is head and neckcancer.

Embodiment III-61

The method of Embodiment III-58, wherein the cancer is prostate cancer.

Embodiment III-62

The method of Embodiment III-58, wherein the cancer is lymphoma.

Embodiment III-63

The method of Embodiment III-58, wherein the cancer is smooth musclecancer.

Embodiment III-64

The method of Embodiment III-58, wherein the cancer is carcinoma of thecolon or rectum.

Embodiment III-65

The method of Embodiment III-58, wherein the cancer is liver cancer.

Embodiment III-66

The method of Embodiment III-58, wherein the cancer is lung cancer.

Embodiment III-67

The method of any of Embodiments III-1-66, further comprisingadministering to the subject in need thereof a therapeutically effectiveamount of one or more additional cancer chemotherapeutic agents inaddition to the platinum drug.

Embodiment III-68

The method of Embodiment III-67, wherein the one or more additionalchemotherapeutic agents is selected from the group consisting of5-fluorouracil, bevacizumab, capecetabine, gemcitabine, irinotecan, andleucovorin.

Embodiment III-69

A pharmaceutical composition formulated for intravenous administrationcomprising

a platinum drug that is cisplatin,

a selective OCT2 inhibitor selected from the group consisting ofbuflomedil, a buflomedil salt, dolutegravir, and a dolutegravir salt,and

a pharmaceutically acceptable carrier.

Embodiment III-70

The pharmaceutical composition of Embodiment III-69, wherein theselective OCT2 inhibitor is buflomedil, or a buflomedil salt.

Embodiment III-71

The pharmaceutical composition of Embodiment III-69, wherein the OCT2inhibitor is dolutegravir, or a dolutegravir salt.

Embodiment III-72

The pharmaceutical composition of any of Embodiments III-69-71, whereinthe amount of the platinum drug is greater than what is present in astandard pharmaceutical composition comprising the platinum drug.

Embodiment III-73

The pharmaceutical composition of any of Embodiments III-69-72, furthercomprising a therapeutically effective amount of one or more additionalcancer chemotherapeutic agents in addition to the platinum drug.

Embodiment III-74

The pharmaceutical composition of Embodiment III-73, wherein the one ormore additional chemotherapeutic agents is selected from the groupconsisting of 5-fluorouracil, bevacizumab, capecetabine, gemcitabine,irinotecan, and leucovorin.

Embodiment III-75

A kit comprising a therapeutically effective amount of a platinum drugthat is cisplatin; a selective OCT2 inhibitor and instructions for use.

Embodiment III-76

The kit of Embodiment III-75, further comprising instructions fordetermining a desirable dose of the selective OCT2 inhibitor for asubject in need.

Embodiment III-77

The kit of Embodiment III-76, wherein the dose is determined based on atleast one factor of the said subject selected from body weight, bodysurface area, height, age, gender, alcohol use, tobacco use, life style,renal function, liver function, genetic polymorphism and co-medications.

Embodiment III-78

The kit of Embodiment III-76, wherein the dose is determined bymonitoring plasma level of the OCT2 inhibitor in the said subject.

Embodiment III-79

The kit of any one of Embodiments III-75-78, wherein the selective OCT2inhibitor is buflomedil or a buflomedil salt.

Embodiment III-80

The kit of Embodiment III-79, wherein buflomedil or a buflomedil salt ispresent in an amount greater than 300 mg, 450 mg, 600 mg, 800 mg or 1000mg.

Embodiment III-81

The kit of any of Embodiments III-75-78, wherein the selective OCT2inhibitor is dolutegravir or a dolutegravir salt.

Embodiment III-82

The kit of Embodiment III-81, wherein dolutegravir or a dolutegravirsalt is present in an amount greater than 50 mg, 75 mg, 100 mg, 150 mg,200 mg or 300 mg.

Embodiment III-83

The kit of any of Embodiments III-75-82, wherein the instructions statethat the kit is intended for use in reducing platinum drug-inducedneurotoxicity.

Embodiment III-84

The kit of any of Embodiments-75-83, wherein the instructions state thatthe kit is intended for use in treating cancer.

Embodiment III-85

The kit of any of Embodiments III-75-84, further comprising atherapeutically effective amount of one or more additional cancerchemotherapeutic agents in addition to the platinum drug.

Embodiment III-86

The kit of Embodiment III-85, wherein the one or more additionalchemotherapeutic agents is selected from the group consisting of5-fluorouracil, bevacizumab, capecetabine, gemcitabine, irinotecan, andleucovorin.

Exemplary Embodiments of Method with Determining Step Embodiment IV-1

In one embodiment, the present disclosure provides a method for reducingplatinum drug-induced toxicity in a subject in need thereof comprising

determining an effective dose to achieve a plasma level of a selectiveOrganic Cation Transporter 2 (OCT2) inhibitor that is buflomedil or abuflomedil salt in the subject in need thereof, wherein the plasma levelis two to five times IC₅₀ of the selective Organic Cation Transporter 2(OCT2) inhibitor that is buflomedil or a buflomedil salt;

administering a platinum drug selected from the group consisting ofoxaliplatin and cisplatin to the subject in need thereof, and

administering an effective dose of a selective Organic CationTransporter 2 (OCT2) inhibitor that is buflomedil or a buflomedil saltto the subject in need thereof,

wherein the toxicity is nephrotoxicity, ototoxicity or peripheralneuropathy, and

wherein the subject in need thereof has a cancer.

Embodiment IV-2

In one embodiment, the present disclosure provides a method for treatingcancer in a subject in need thereof comprising

determining an effective dose to achieve a plasma level of a selectiveOrganic Cation Transporter 2 (OCT2) inhibitor that is buflomedil or abuflomedil salt in the subject in need thereof, wherein the plasma levelis two to five times IC₅₀ of the selective Organic Cation Transporter 2(OCT2) inhibitor that is buflomedil or a buflomedil salt;

administering a therapeutically effective amount of a platinum drugselected from the group consisting of oxaliplatin and cisplatin to thesubject in need thereof; and

administering an effective dose of a selective Organic CationTransporter 2 (OCT2) inhibitor that is buflomedil or a buflomedil saltto the subject in need thereof.

Embodiment IV-3

In one embodiment, the present disclosure provides a method forincreasing patient compliance for treating cancer in a subject in needthereof comprising

determining an effective dose to achieve a plasma level of a selectiveOrganic Cation Transporter 2 (OCT2) inhibitor that is buflomedil or abuflomedil salt in the subject in need thereof, wherein the plasma levelis two to five times IC₅₀ of the selective Organic Cation Transporter 2(OCT2) inhibitor that is buflomedil or a buflomedil salt;

administering a therapeutically effective amount of a platinum drugselected from the group consisting of oxaliplatin and cisplatin to thesubject in need thereof; and

administering a selective Organic Cation Transporter 2 (OCT2) inhibitorthat is buflomedil or a buflomedil salt to the subject in need thereofin a dose effective to reduce platinum drug-induced toxicity in saidsubject in need thereof, whereby said subject completes treatment with acumulative dose of at least 100 mg/m².

Embodiment IV-4

The method of any one of Embodiments IV-1-3, wherein the step ofdetermining an effective dose to achieve a plasma level of a selectiveOrganic Cation Transporter 2 (OCT2) inhibitor is performed byadministering a pre-dose of the selective Organic Cation Transporter 2(OCT2) inhibitor.

Embodiment IV-5

The method of any one of Embodiments IV-1-3, wherein the step ofdetermining an effective dose to achieve a plasma level of a selectiveOrganic Cation Transporter 2 (OCT2) inhibitor is determined by areference data chart based on subject characteristics.

Embodiment IV-6

The method of any one of Embodiments IV-2 or 3-5, wherein thetherapeutically effective amount of platinum drug administered is knownto cause platinum drug-induced neurotoxicity in subjects.

Embodiment IV-7

The method of Embodiment IV-6, wherein the neurotoxicity is peripheralneurotoxicity.

Embodiment IV-8

The method of Embodiment IV-6, wherein the neurotoxicity is damage to asensory neuron.

Embodiment IV-9

The method of Embodiment IV-6, wherein the neurotoxicity is damage to amotor neuron.

Embodiment IV-9a

The method of any of Embodiments IV-1-9, wherein the neurotoxicity ischronic neurotoxicity.

Embodiment IV-9b

The method of any of Embodiments IV-1-9, wherein the neurotoxicity isacute syndrome transient neurotoxicity.

Embodiment IV-9c

The method of any of Embodiments IV-1-9, wherein the neurotoxicityoccurs 1 hour to seven days after first treatment.

Embodiment IV-9d

The method of any of Embodiments II-1-9, wherein the neurotoxicityoccurs after a subject completes treatment with a cumulative dose of atleast 500 mg/m²

Embodiment IV-10

The method of Embodiment IV-6, wherein the neurotoxicity is damage todorsal root ganglia (DRG).

Embodiment IV-11

The method of any of Embodiments IV-1-10, wherein the dose of selectiveOCT2 inhibitor is effective to minimize platinum drug-inducedneurotoxicity in said subject in need thereof.

Embodiment IV-12

The method of any of Embodiments IV-1-10, wherein the dose of selectiveOCT2 inhibitor is effective to prevent platinum drug-inducedneurotoxicity of Grade 3 or higher, as assessed by a method selectedfrom National Cancer Institute-Common Toxicity Criteria (NCI-CTC)sensory scale, National Cancer Institute-Common Toxicity Criteria(NCI-CTC) motor scale, Total Neuropathy Score clinical version (TNSc)and European Organization for Research and Treatment of Cancer CIPNspecific self-report questionnaire (EORTC QOL-CIPN20), in patientstreated with a cumulative dose of at least 100 mg/m².

Embodiment IV-13

The method of any of Embodiments IV-1-10, wherein the dose of selectiveOCT2 inhibitor is effective to prevent platinum drug-inducedneurotoxicity of Grade 3 or higher, as assessed by a method selectedfrom National Cancer Institute-Common Toxicity Criteria (NCI-CTC)sensory scale, National Cancer Institute-Common Toxicity Criteria(NCI-CTC) motor scale, Total Neuropathy Score clinical version (TNSc)and European Organization for Research and Treatment of Cancer CIPNspecific self-report questionnaire (EORTC QOL-CIPN20), in patientstreated with a platinum drug dose intensity of at least 30 mg/week/m².

Embodiment IV-14

The method of any of Embodiments IV-1-10, wherein the dose of selectiveOCT2 inhibitor is effective to prevent platinum drug-inducedneurotoxicity of Grade 3 or higher, as assessed by a method selectedfrom National Cancer Institute-Common Toxicity Criteria (NCI-CTC)sensory scale, National Cancer Institute-Common Toxicity Criteria(NCI-CTC) motor scale, Total Neuropathy Score clinical version (TNSc)and European Organization for Research and Treatment of Cancer CIPNspecific self-report questionnaire (EORTC QOL-CIPN20), in patientstreated with a platinum drug dose of at least 80 mg/m².

Embodiment IV-15

The method of any of Embodiments IV-1-14, further comprising assessingplatinum drug-induced neurotoxicity in a subject after administration ofthe platinum drug.

Embodiment IV-16

The method of Embodiment IV-15, wherein the neurotoxicity is assessed bya method selected from National Cancer Institute-Common ToxicityCriteria (NCI-CTC) sensory scale, National Cancer Institute-CommonToxicity Criteria (NCI-CTC) motor scale, Total Neuropathy Score clinicalversion (TNSc) and European Organization for Research and Treatment ofCancer CIPN specific self-report questionnaire (EORTC QOL-CIPN20).

Embodiment IV-17

The method of Embodiment IV-15, wherein the neurotoxicity is assessed bya measurement selected from sensory nerve action potential, sensorynerve conduction velocity, cold pain threshold, heat pain threshold,mechanical pain threshold, cold detection threshold, warm detectionthreshold, mechanical detection threshold, vibration perceptionthreshold, current perception threshold, pinprick sensibility, deeptendon reflexes and grip strength.

Embodiment IV-18

The method of Embodiment IV-15, wherein the neurotoxicity is assessed bymeasuring sensory nerve action potential in one of radial, dorsal sural,sural and ulnar nerves.

Embodiment IV-19

The method of any of Embodiments IV-15-18, further compromisingestablishing the subject's baseline prior to administration of theplatinum drug.

Embodiment IV-20

The method of any of Embodiments IV-15-18, wherein the platinumdrug-induced neurotoxicity is assessed near the midpoint of treatment.

Embodiment IV-21

The method of any of Embodiments IV-15-18, wherein the platinumdrug-induced neurotoxicity is assessed after treatment with a cumulativedose of 200 mg/m².

Embodiment IV-22

The method of Embodiment IV-1, wherein toxicity is nephrotoxicity.

Embodiment IV-23

The method of any of Embodiments IV-1-22, wherein the dose of buflomedilor a buflomedil salt administered in a subject is adjusted based on atleast one of the factors of the said subject, body weight, body surfacearea, height, age, gender, alcohol use, tobacco use, life style, renalfunction, liver function, genetic polymorphism and co-medications.

Embodiment IV-24

The method of any of Embodiments IV-1-23, wherein the dose of buflomedilor a buflomedil salt results in its plasma level during the period ofplatinum administration at least 0.43 mg/l, 0.86 mg/l, 1.29 mg/l, 1.72mg/l or 2.15 mg/l.

Embodiment IV-25

The method of any of Embodiments IV-1-24, wherein the dose of buflomedilor a buflomedil salt is at least 100 mg, 200 mg, 300 mg, 400 mg, 500 mgor 600 mg.

Embodiment IV-26

The method of any of Embodiments IV-1-25, wherein the dose of buflomedilor a buflomedil salt is at least 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5mg/kg, 6 mg/kg, 8 mg/kg or 10 mg/kg.

Embodiment IV-27

The method of any of Embodiments IV-1-26, wherein the selective OCT2inhibitor does not reduce the efficacy of the platinum drug.

Embodiment IV-28

The method of any of Embodiments IV-1-27, wherein the selective OCT2inhibitor is administered at a dose that results in its plasmaconcentration during the period of platinum drug administration lessthan its maximum tolerated plasma concentration (MTC) and greater than1×, 2×, 3×, 4× of its IC₅₀ value for OCT2-mediated transport of 20 μMoxaliplatin assessed in human serum or an assay buffer containing 4%bovine serum albumin.

Embodiment IV-29

The method of any of Embodiments IV-1-28, wherein the selective OCT2inhibitor has an IC₅₀ for OCT2-mediated transport of 20 μM oxaliplatinin human serum or an assay solution containing 4% bovine serum albumin,of less than or equal to 5 μM.

Embodiment IV-30

The method of Embodiment IV-29, wherein the selective OCT2 inhibitor hasan IC₅₀ for OCT2-mediated transport of 20 μM oxaliplatin in human serumor an assay solution containing 4% bovine serum albumin that is at least10-fold less than the selective OCT2 inhibitor IC₅₀ for 20 μMoxaliplatin transport in human serum or an assay solution containing 4%bovine serum albumin mediated by one or more of OCT1-, OCT-3, or MATE-1.

Embodiment IV-31

The method of any of Embodiments IV-1-30, wherein the subject in needthereof has a cancer expressing at least one of Organic CationTransporter 1 (OCT1) or Organic Cation Transporter 3 (OCT3).

Embodiment IV-32

The method of any of Embodiments IV-1-31, wherein the platinum drug andthe selective OCT2 inhibitor are administered at the same time.

Embodiment IV-33

The method of any of Embodiments IV-1-31, wherein the selective OCT2inhibitor is administered before the platinum drug.

Embodiment IV-34

The method of any of Embodiments IV-1-31, wherein the selective OCT2inhibitor is administered after the platinum drug.

Embodiment IV-35

The method of any of Embodiments IV-1-34, wherein the amount of platinumdrug administered to the subject during one treatment session is greaterthan what is administered under standard clinical practices.

Embodiment IV-36

The method of any of Embodiments IV-1-35, wherein the cumulative amountof platinum drug administered to the subject in need thereof over theentire course of treatment is greater than what is administered understandard clinical practices.

Embodiment IV-37

The method of any of Embodiments IV-1-36, wherein the platinum drug isadministered at a greater frequency than under standard clinicalpractices.

Embodiment IV-38

The method of any of Embodiments IV-1 or 3-37, wherein the neurotoxicityis peripheral neuropathy.

Embodiment IV-39

The method of Embodiment IV-38, wherein the peripheral neuropathy isGrade 3 or Grade 4 peripheral neuropathy.

Embodiment IV-40

The method of any of Embodiments IV-1-39, wherein the subject in needthereof is a human or a non-human animal.

Embodiment IV-41

The method of any of Embodiments IV-1-40, wherein the selective OCT2inhibitor is administered enterally, intravenously, intramuscularly,intraperitoneally, orally, or parenterally.

Embodiment IV-42

The method of any of Embodiments IV-1-41, wherein the selective OCT2inhibitor is administered via more than one route of administration.

Embodiment IV-43

The method of any of Embodiments IV-1-42, wherein the platinum drug andthe selective OCT2 inhibitor are administered via the same route ofadministration.

Embodiment IV-44

The method of any of Embodiments IV-1-43, wherein the selective OCT2inhibitor is administered via intravenous infusion.

Embodiment IV-45

The method of any of Embodiments IV-1-43, wherein the selective OCT2inhibitor is administrated via intravenous injection and intravenousinfusion.

Embodiment IV-46

The method of Embodiment IV-44 or 45, wherein intravenous infusion isover a period of time at least 1 hour.

Embodiment IV-47

The method of Embodiment IV-44 or 45, wherein the rate of intravenousinfusion is constant.

Embodiment IV-48

The method of Embodiments IV-44 or 45, wherein the rate of intravenousinfusion is variable.

Embodiment IV-49

The method of any of Embodiments IV-1-48, wherein the cancer expressesOCT1.

Embodiment IV-50

The method of any of Embodiments IV-1-49, wherein the cancer expressesOCT3.

Embodiment IV-51

The method of any of Embodiments IV-1-50, wherein the cancer expressesOCT1 and OCT3.

Embodiment IV-52

The method of any one of Embodiments IV-1-51, wherein the cancer isselected from the group consisting of adenocarcinoma of the pancreas,ampullary and periampullary carcinoma, adenocarcinoma of the anus,appendiceal carcinoma, hepatocellular carcinoma, carcinoma of the colonor rectum, epithelial ovarian carcinoma, fallopian tube carcinoma.primary peritoneal cancer, esophageal or esophagogastric junctioncarcinoma, gastric carcinoma, small bowel carcinoma, testicular cancer,cholangiocarcinoma, pancreatic adenocarcinoma, carcinoma of unknownprimary origin, chronic lymphocytic leukemia/small lymphocytic lymphoma,non-Hodgkin's lymphoma, adult T-cell leukemia/lymphoma, AIDS-relatedB-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma,gastric MALT lymphoma, nongastric MALT lymphoma, mantle cell lymphoma,mycosis fungoides/Sezary syndrome, splenic marginal zone lymphoma,peripheral T cell lymphoma, primary cutaneous B-cell lymphoma, primarycutaneous anaplastic large cell lymphoma (ALCL), lung cancer, livercancer, head and neck cancer, prostate cancer, smooth muscle cancer andgallbladder cancer.

Embodiment IV-53

The method of Embodiment IV-52, wherein the cancer is ovarian cancer.

Embodiment IV-54

The method of Embodiment IV-52, wherein the cancer is head and neckcancer.

Embodiment IV-55

The method of Embodiment IV-52, wherein the cancer is prostate cancer.

Embodiment IV-56

The method of Embodiment IV-52, wherein the cancer is lymphoma.

Embodiment IV-57

The method of Embodiment IV-52, wherein the cancer is smooth musclecancer.

Embodiment IV-58

The method of Embodiment IV-52, wherein the cancer is carcinoma of thecolon or rectum.

Embodiment IV-59

The method of Embodiment IV-52, wherein the cancer is liver cancer.

Embodiment IV-60

The method of Embodiment IV-52, wherein the cancer is lung cancer.

Embodiment IV-61

The method of any of Embodiments IV-1-60, further comprisingadministering to the subject in need thereof a therapeutically effectiveamount of one or more additional cancer chemotherapeutic agents inaddition to the platinum drug.

Embodiment IV-62

The method of Embodiment IV-61, wherein the one or more additionalchemotherapeutic agents is selected from the group consisting of5-fluorouracil, bevacizumab, capecetabine, gemcitabine, irinotecan, andleucovorin.

Embodiment IV-63

A pharmaceutical composition formulated for intravenous administrationcomprising

a platinum drug that is oxaliplatin or cisplatin,

a selective OCT2 inhibitor selected from the group consisting ofbuflomedil and a buflomedil salt, and

a pharmaceutically acceptable carrier.

Embodiment IV-64

The pharmaceutical composition of Embodiment IV-63, wherein the amountof the platinum drug is greater than what is present in a standardpharmaceutical composition comprising the platinum drug.

Embodiment IV-65

The pharmaceutical composition of Embodiment IV-63 or 64, furthercomprising a therapeutically effective amount of one or more additionalcancer chemotherapeutic agents in addition to the platinum drug.

Embodiment IV-66

The pharmaceutical composition of Embodiment IV-65, wherein the one ormore additional chemotherapeutic agents is selected from the groupconsisting of 5-fluorouracil, bevacizumab, capecetabine, gemcitabine,irinotecan, and leucovorin.

Embodiment IV-67

A kit comprising a therapeutically effective amount of a platinum drugthat is oxaliplatin or cisplatin; a selective OCT2 inhibitor andinstructions for use.

Embodiment IV-68

The kit of Embodiment IV-67, further comprising instructions fordetermining a desirable dose of the selective OCT2 inhibitor for asubject in need.

Embodiment IV-69

The kit of Embodiment IV-67 or 68, wherein the dose is determined basedon at least one factor of the said subject selected from body weight,body surface area, height, age, gender, alcohol use, tobacco use, lifestyle, renal function, liver function, genetic polymorphism andco-medications.

Embodiment IV-70

The kit of any of Embodiments IV-67-69, wherein the dose is determinedby monitoring plasma level of the OCT2 inhibitor in the said subject.

Embodiment IV-71

The kit of any of Embodiments IV-67-70, wherein buflomedil or abuflomedil salt is present in an amount greater than 300 mg, 450 mg, 600mg, 800 mg or 1000 mg.

Embodiment IV-72

The kit of any of Embodiments IV-67-71, wherein the instructions statethat the kit is intended for use in reducing platinum drug-inducedneurotoxicity.

Embodiment IV-73

The kit of Embodiment IV-67-72, wherein the instructions state that thekit is intended for use in treating cancer.

Embodiment IV-74

The kit of any of Embodiments IV-67-73 further comprising atherapeutically effective amount of one or more additional cancerchemotherapeutic agents in addition to the platinum drug.

Embodiment IV-75

The kit of Embodiment IV-74, wherein the one or more additionalchemotherapeutic agents is selected from the group consisting of5-fluorouracil, bevacizumab, capecetabine, gemcitabine, irinotecan, andleucovorin.

Exemplary Embodiments-Selective OCT2 Inhibitor for Use in TreatmentEmbodiment V-1

In one embodiment, the present disclosure provides a selective OCT2inhibitor for use in the treatment of cancer in a subject in needthereof, wherein the OCT2 inhibitor is for use in combination with aplatinum drug that is oxaliplatin.

Embodiment V-2

The selective OCT2 inhibitor according to Embodiment V-1, wherein theOCT2 inhibitor is for administration at a dose capable of reducingplatinum drug-induced toxicity.

Embodiment V-3

In one embodiment, the present disclosure provides a selective OCT2inhibitor for use in the treatment of cancer in a subject in needthereof, wherein the OCT2 inhibitor is for use in combination with aplatinum drug that is cisplatin.

Embodiment V-4

The selective OCT2 inhibitor according to Embodiment V-3, wherein theOCT2 inhibitor is for administration at a dose capable of reducingplatinum drug-induced toxicity.

Embodiment V-5

In one embodiment, the present disclosure provides a selective OCT2inhibitor that is buflomedil or a buflomedil salt for use in thetreatment of cancer in a subject in need thereof, wherein the OCT2inhibitor is for use in combination with a platinum drug that isoxaliplatin or cisplatin.

Embodiment V-6

The selective OCT2 inhibitor according to Embodiment V-5, wherein theOCT2 inhibitor is for administration at a dose capable of reducingplatinum drug-induced toxicity.

Embodiment V-7

In one embodiment, the present disclosure provides a selective OCT2inhibitor for use in reducing platinum drug toxicity in a subject inneed thereof, wherein the OCT2 inhibitor is for use in combination witha platinum drug that is oxaliplatin.

Embodiment V-8

In one embodiment, the present disclosure provides a selective OCT2inhibitor for use in reducing platinum drug toxicity in a subject inneed thereof, wherein the OCT2 inhibitor is for use in combination witha platinum drug that is cisplatin.

Embodiment V-9

In one embodiment, the present disclosure provides a selective OCT2inhibitor that is buflomedil or a buflomedil salt for use in reducingplatinum drug toxicity in a subject in need thereof, wherein the OCT2inhibitor is for use in combination with a platinum drug that isoxaliplatin or cisplatin.

Embodiment V-10

In one embodiment, the present disclosure provides a selective OCT2inhibitor for use in increasing patient compliance in the treatment ofcancer in a subject in need thereof, wherein the OCT2 inhibitor is foruse in combination with a platinum drug that is oxaliplatin.

Embodiment V-11

In one embodiment, the present disclosure provides a selective OCT2inhibitor for use in increasing patient compliance in the treatment ofcancer in a subject in need thereof, wherein the OCT2 inhibitor is foruse in combination with a platinum drug that is cisplatin.

Embodiment V-12

In one embodiment, the present disclosure provides a selective OCT2inhibitor that is buflomedil or a buflomedil salt for use in increasingpatient compliance in the treatment of cancer in a subject in needthereof, wherein the OCT2 inhibitor is for use in combination with aplatinum drug that is oxaliplatin or cisplatin.

Exemplary Embodiments—Use of Selective OCT2 Inhibitor Embodiment VI-1

In one embodiment, the present disclosure provides use of a selectiveOCT2 inhibitor in the preparation of a medicament for the treatment ofcancer, wherein the medicament is for use in combination with a platinumdrug that is oxaliplatin.

Embodiment VI-2

The use according to Embodiment VI-1, wherein the OCT2 inhibitor is foradministration at a dose capable of reducing platinum drug-inducedtoxicity.

Embodiment VI-3

In one embodiment, the present disclosure provides use of a selectiveOCT2 inhibitor in the preparation of a medicament for the treatment ofcancer, wherein the medicament is for use in combination with a platinumdrug that is cisplatin.

Embodiment VI-4

The use according to Embodiment VI-3, wherein the OCT2 inhibitor is foradministration at a dose capable of reducing platinum drug-inducedtoxicity.

Embodiment VI-5

In one embodiment, the present disclosure provides use of a selectiveOCT2 inhibitor that is buflomedil or a buflomedil salt in thepreparation of a medicament for the treatment of cancer, wherein themedicament is for use in combination with a platinum drug that isoxaliplatin or cisplatin.

Embodiment VI-6

The use according to Embodiment VI-5, wherein the OCT2 inhibitor is foradministration at a dose capable of reducing platinum drug-inducedtoxicity.

Embodiment VI-7

In one embodiment, the present disclosure provides use of a selectiveOCT2 inhibitor in the preparation of a medicament for reducing platinumdrug toxicity, wherein the medicament is for use in combination with aplatinum drug that is oxaliplatin.

Embodiment VI-8

In one embodiment, the present disclosure provides use of a selectiveOCT2 inhibitor in the preparation of a medicament for reducing platinumdrug toxicity, wherein the medicament is for use in combination with aplatinum drug that is cisplatin.

Embodiment VI-9

In one embodiment, the present disclosure provides use of a selectiveOCT2 inhibitor that is buflomedil or a buflomedil salt in thepreparation of a medicament for reducing platinum drug toxicity, whereinthe medicament is for use in combination with a platinum drug that isoxaliplatin or cisplatin.

Embodiment VI-10

In one embodiment, the present disclosure provides use of a selectiveOCT2 inhibitor in the preparation of a medicament for increasing patientcompliance in the treatment of cancer, wherein the medicament is for usein combination with a platinum drug that is oxaliplatin.

Embodiment VI-11

In one embodiment, the present disclosure provides use of a selectiveOCT2 inhibitor in the preparation of a medicament for increasing patientcompliance in the treatment of cancer, wherein the medicament is for usein combination with a platinum drug that is cisplatin.

Embodiment VI-12

In one embodiment, the present disclosure provides use of a selectiveOCT2 inhibitor that is buflomedil or a buflomedil salt in thepreparation of a medicament for increasing patient compliance in thetreatment of cancer, wherein the medicament is for use in combinationwith a platinum drug that is oxaliplatin or cisplatin.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

It understood that Embodiments V-1 to V-6 and VI-1 to VI-6 can be usedin combination with any of Embodiments I-1 to I-105; II-1 to II-85;III-1 to III-86; or IV-1 to IV-75.

EXAMPLES

The following Examples are merely illustrative and are not meant tolimit any aspects of the present disclosure in any way.

Example A-Protocol for Evaluation of the Protective Effect of OPV-3003in a Balb/c Mice Model of Oxaliplatin-Induced Peripheral Neuropathy

The protocol in this example is discussed in Renn, C. L.; Carozzi, V.A.; Rhee, P.; Gallop, D.; Dorsey, S. G.; Cavaletti, G., Multimodalassessment of painful peripheral neuropathy induced by chronicoxaliplatin-based chemotherapy in mice. Mol Pain, 2011, 7, 29; andBoehmerle, W., et. al., Scientific Reports 4:6370, p 1-9, 2014.

Aim

The aim is to assess the neuroprotective effects of OPV-3003 in Balb/cmice exposed to chronic treatment with Oxaliplatin (OHP).

Animals

Male Balb/c mice, 20 grams on arrival (Envigo, Italy)

Housing and Treatments

The care and husbandry of animals were in conformity with theinstitutional guidelines in compliance with national (D.L. n. 26/2014)and international laws and policies (EEC Council Directive 86/609, OJ L358, 1, Dec. 12, 1987; Guide for the Care and Use of Laboratory Animals,U.S. National Research Council, 1996).

Drugs

OXALIPLATIN (OHP), 3.5 mg/kg, intravenously, twice a week for 4 weeks.

OPV-3003 (OPV), 30 mg/kg intraperitoneally 15 minutes before OHPadministration, and 15 mg/kg intravenously co-administrated with OHP,twice a week for 4 weeks.

Experimental Protocol Randomization

GROUP 1: NAÏVE (N=12)

GROUP 2: OPV (N=12)

GROUP 3: OHP (N=12)

GROUP 4: OHP+OPV (n=12)

Total number of animals: 48

As shown in the flow-chart in FIG. 18:

-   -   at baseline, at the end of treatment and at the end of follow-up        period nerve conduction velocity and behavioral test will be        performed in all the animals;    -   15 min after the first and the last iv administration the serum        sample will be collected for pharmacokinetic analysis;    -   24 h after the first iv administration the cold plate test will        be performed;    -   at the end of treatment and at the end of follow-up period        sciatic nerve, caudal nerve, L4-L5 DRG and skin biopsies will be        collected by 4 animals groups;    -   the body weight will be measured twice a week; mortality and        clinical signs will be monitored every day.

Experimental Plan

Phase 1: Pharmacological Treatments

OHP and OPV will be administered for 4 weeks. In the groupco-administered with OHP and OPV, OPV intraperitoneally will be injected15 min before the co-administration of OHP and OPV intravenously.

4 animals in each group will be sacrificed four days after the last drugadministration.

Phase 2: Follow-Up Period

The animals will be observed for 4 and 8 weeks after the lastadministration. After 4 weeks of follow-up the analysis of NCV will beperformed to decide if sacrifice the animals or observe the animal forother 4 weeks.

Assessments and Timing Nerve Conduction Velocity

The caudal and digital NCV will be evaluated at baseline, at the end ofthe pharmacological treatment and after 4 and 8 weeks of follow-up.Sensory/motor and sensory nerve conduction velocities (NCVs) aredetermined stimulating respectively the caudal and the digital nerves byusing an electromyography apparatus (Myto2 ABN Neuro, Firenze, Italy).The caudal NCV will be determined by placing a couple of recordingneedle electrodes at the base of the tail and a couple of stimulatingneedle electrodes 3.5 cm distally to the recording points. The digitalNCV will be determined by placing the positive recording electrode inthe thigh, the negative recording electrode close to ankle bone and thepositive and negative stimulating electrodes close to the fourth toenear the digital nerve and under the paw respectively.

The intensity, duration and frequency of stimulation will be set up inorder to obtain optimal results.

All the neurophysiological determinations will be performed understandard conditions in a temperature-controlled room (22+/−2° C.).

Behavioral Test

The Plantar Test, Dynamic Plantar Aesthesiometer Test and Cold PlateTest will be used to determine the alterations in pain perception andtheir changes due to pharmacological treatment. These behavioral testswill be performed at baseline, at the end of pharmacological treatmentand after 4 and 8 weeks of follow-up.

The thermal nociceptive threshold of the plantar rear paw will beassessed using a Plantar Test (model 37370; Ugo Basile BiologicalInstruments, Comerio, Italy). An infra-red light source is located underthe glass floor and positioned under the center of the mice's rear paw.Once in position, the heat source was activated and a photo cellautomatically shut off the heat source and recorded the time towithdrawal (withdrawal latency). To avoid causing thermal injury in theevent that the animal did not withdraw its paw voluntarily, there was anupper limit cutoff of 30 seconds, after which the heat was automaticallyterminated.

The mechanical nociceptive threshold will be assessed using a DynamicAesthesiometer Test (model 37450, Ugo Basile Biological Instruments,Comerio, Italy), which generated a linearly increasing mechanical force.

At each time point, after the acclimatization period, a pointed metallicfilament (0.5-mm diameter) will be applied to the plantar surface of thehind paw, which exerted a progressively increasing punctuate pressure,reaching up to 15 g within 15 seconds. The pressure evoking a clearvoluntary hind-paw withdrawal response will be recorded automaticallyand taken as representing the mechanical nociceptive threshold index.The results represent the maximal pressure (expressed in grams)tolerated by the animals. There was an upper limit cutoff of 30 seconds,after which the mechanical stimulus was automatically terminated.

Cold plate test is performed by using an apparatus (35100—Hot/ColdPlate, Ugo Basile instruments) composed by a Plexiglas cylinder and athermostatic plate, able to reach variable temperatures. The animal isplaced on the plate set at +4° C., free to move and walk. Two blindedexperimenters simultaneously determine the number of pain signs (ex:jumping, licking, increase anxiety etc . . . ) in a trial of 5 minutes.

Morphological Analysis of DRG, Sciatic and Caudal Nerves

Left sciatic nerves, caudal nerves, L4-L5 DRG, will be harvested andprocessed for the morphological analysis. The tissues will be fixed for3 hours at room temperature in paraformaldehyde 4%/glutaraldehyde 2%(DRG) or in glutaraldehyde 3% (peripheral nerves), post-fixed in OSO₄and epoxy resin embedded. Morphological analysis will be carried out on1 μm-thick semi-thin sections stained with toluidine blue. At least twotissue blocks for each animal will be sectioned and then examined with alight microscope.

Morphometrical Analysis of DRG and Nerves

Harvested DRG will be used for the morphometric examinations ontoluidine blue stained 1-μm-thick semithin sections. DRG will beanalyzed with a computer-assisted image analyzer (ImageJ NIH software)and the somatic, nuclear and nucleolar size of DRG sensory neuronsmeasured in randomly selected sections on at least 300 DRGneurons/mouse.

For the morphometric analysis of myelinated fibers, sections will beobserved with a photomicroscope (Nikon Eclipse E200; Leica MicrosystemsGmbH, Wetzlar, Germany) at a magnification of 60× and the morphometricanalysis was performed using a QWin automatic image analyzer (LeicaMicrosystems GmbH). In randomly selected sections collected from allspecimens, all myelinated fibers evaluable in the analyzed space will becounted and the external (total) and internal (axonal) diameters ofmyelinated fibers will be measured, on at least 500 myelinatedfibers/nerves. From both axonal and total fiber diameters, the histogramof fiber distribution will be calculated and the ratio between the twodiameters, the g-ratio (a well-established measure of degree ofmyelination [Boehmerle 2014]), will be automatically calculated for eachset of individual axon and fiber diameter.

Example 1

The following example relates to the identification of inhibitors ofoxaliplatin cellular uptake through the OCT2 transporter.

Oxaliplatin is among the most actively used tumor chemotherapeutics,particularly in colorectal cancer. However, along with its antitumorefficacy, oxaliplatin has been associated with adverse effects includingperipheral neuropathy, ototoxicity and nephrotoxicity. Although theexact mechanism of these toxicities remains poorly understood, it hasbeen proposed that OCT2 mediated cellular accumulation may be a cause[1]. A library of prescription drugs against oxaliplatin uptake inOCT2-expressing cells was screened and a number of drugs as inhibitorsof OCT2-mediated oxaliplatin cellular uptake were identified.

Materials and Methods

Madin-Darby canine kidney type II (MDCK-II) cells maintained inDulbecco's Modified Eagle's Medium (DMEM) were seeded at 60±10K per wellon 96-well PCF porous membrane insert 24 h prior to transfection. Fullyconfluent MDCK cell monolayers were transfected with either cDNAplasmids encoding the human transporter to be tested or a GFP control. Atransport assay was initiated 48 h later by incubating cells with theprobe substrate mixed with varying concentrations of the test drug at37° C., for 5 or 30 min. The cells were then washed with ice-cold PBSand lysed with 50:50 acetonitrile:H2O. Intracellular content of thesubstrate was quantified by LC/MS/MS or a MicroBeta radiometric platereader.

Fifteen drugs were selected and evaluated for their potencies ininhibiting OCT2-mediated oxaliplatin uptake at 5 and 50 μM to obtainestimated IC₅₀ values (Table 1).

TABLE 1 Inhibition profile of drags with respect to OCT2-mediatedsubstrate uptake Substrate used Oxaliplatin ASP⁺ Metformin InhibitionInhibition Inhibition Inhibition Inhibition % @ % @ IC₅₀ % @ IC₅₀ % @ %@ IC₅₀ Compound 5 μM 50 μM (μM) ** 20 μM * (μM) ** 5 μM 50 μM (μM) **Buflomedil 87 105 0.78 104 <0.1 5.7 ± 0.8 Chlorphenesin 14 30 117.7 960.86 no inhibition at 100 uM Miconazole 39 97 1.55 91 2.0 141.6 ± 50.2 Leuprolide 76 97 1.57 88 2.8 58 91  3.62 Erlotinib 93 106 0.37 Unkn 94.4± 18.0 Cimetidine 34 83 9.7 43 26.4 57.6 ± 6.9  Chlorphenesin 28 36 90.4Unkn no Carbamate inhibition at 100 uM Ipratropium-Br 107 104 <0.1 960.81 94 97  0.32 Propantheline-Br 103 102 <0.1 107 <0.1 75 96 1.7Tiotropium-Br 98 102 0.12 Unkn 77 95 1.5 Dolutagravir*** 83 97 0.20 Unkn0.54 ± 0.09 Exemestane 96 102 0.19 100 <0.1 66 94 2.6 Epinastine 96 1000.19 97 0.58 56 92 3.9 Pentamidine 99 99 0.07 102 <0.1 67 91 2.5Imatinib 57 95 3.7 103 <0.1 ASP+ was used at 5 μM in Hanks' BalancedSalt Solution (HBSS). Meformin and oxaliplatin were dosed at 10 μM inHBSS for 5 and 30 min, respectively. ASP+:4-(4-(Dimethylamino)styryl)-N-methylpyridinium iodide Unkn: unknown *Kido Y et al. J Med Chem 2011; 54 (13): 4548-58. ** IC₅₀ is estimatedbased on the inhibition induced by the single concentration (20 or 50μM) tested, or calculated by nonlinear regression of sevenconcentrations tested (in bold, ±SEM). ***IC₅₀ values were adjusted withmeasured inhibitor recovery rate.

Many prescription drugs have been screened against human OCT2 expressedin various cell lines using different OCT2 substrates. For example,using ASP⁺ as an OCT2 substrate, the Giacomini group [2] identified 244prescription drugs as OCT2 inhibitors, some of which are listed inTable 1. However, the extent to which various inhibitors reducedOCT2-mediated transport of other substrates, such as metformin andoxaliplatin, varies widely, possibly due to the fact that thesesubstrates interact with distinct binding sites of OCT2 [3]. Thus thereexists no direct correlation between the inhibition potencies ofOCT2-mediated ASP⁺ uptake and OCT2-mediated uptake of oxaliplatin andmetformin. For example, chlorphenesin carbamate and imatinib were shownby the Giacomini group to be a potent inhibitor of OCT2-mediated ASP⁺uptake, but their potencies as an inhibitor of OCT2-mediated metforminand oxaliplatin uptake are more than 100 and 30 fold less, respectively.In another example, miconazole exhibited similar potency against OCT2transport of ASP⁺ and oxaliplatin, but is approximately 70 fold lesspotent in inhibiting OCT2-mediated metformin transport (Table 1).

Metformin is a more commonly used probe substrate for OCT2. Theinhibition of metformin transport through OCT2 was also not predictiveof a drug's effect on OCT2-mediated oxaliplatin uptake. Applicantsscreened 70 compounds for OCT2-mediated transport of metformin. Forexample, miconazole, erlotinib, pentamidine, epinastine, exemestrane,tiotropium and propantheline demonstrated over 10-fold higher potenciesin inhibiting OCT2-mediated transport of oxaliplatin than that ofmetformin (Table 1). For example, leuprolide and miconazole demonstratedabout 39-fold differences in metformin transport (IC₅₀=3.62 and 141.60μM), but displayed similar potency in inhibiting oxaliplatin uptake(IC₅₀=1.57 and 1.55 μM, respectively).

The data indicated that in order to determine the inhibition potency ofdrugs against OCT2-mediated oxaliplatin transport, oxaliplatin must beused as the OCT2 substrate. Therefore, previously reported studies [2,3]based on in vitro OCT2 assays using substrates other than oxaliplatincannot be used to determine whether compounds inhibit OCT2-mediatedoxaliplatin transport.

Using oxaliplatin as the OCT2 substrate, a number of drugs wereidentified, including buflomedil, as potent inhibitors (IC₅₀<2 μM) ofOCT2-mediated oxaliplatin transport. Under in vivo conditions,non-specific binding of inhibitors to serum proteins can affect theirapparent inhibition potencies in blood plasma. To further confirm theirpotencies in blood plasma, which are reflected by IC_(50,app), serialconcentrations of these drugs were tested against 20 μM or 100 Moxaliplatin transport mediated through OCT2 in either 100% human serumor HBSS containing 4% bovine serum albumin (BSA), which closelyrepresents serum in terms of protein binding of drugs. The regressedIC₅₀ and apparent serum IC_(50,app) values of part of the drugs areshown in Table 2.

TABLE 2 Identification of clinically relevant inhibitors of OCT2mediated oxaliplatin uptake by C_(max, u)/IC₅₀ or C_(max)/IC_(50, app)ratio Plasma peak Plasma peak total Plasma unbound IC₅₀ or KiIC_(50, app) concentration protein concentration Compound (μM) * (μM) **C_(max) (μM) binding (%) C_(max, u) (μM) C_(max, u)/IC₅₀C_(max)/IC_(50, app) Buflomedil 0.78 1.45 6.8 70 2.04 2.62 4.69Miconazole 1.55 3.30 14.9 92 1.19 0.77 4.52 Erlotinib 0.37 2.08 5.4 930.38 1.02 2.60 Propantheline-Br <0.1 0.24 0.134 unkn 0.56 Epinastine0.19 0.12 64.2 0.04 0.23 Cimetidine 9.71 36.75 12 20 9.60 0.99 0.33Tiotropium Br 0.12 1.10 0.034 72 0.01 0.08 0.03 Exemestane 0.19 0.091 900.01 0.05 Pentamidine 0.067 0.730 69 0.23 3.38 Dolutegravir 0.20 3.3710.90 99.5 0.05 0.28 3.23 Leuprolide 1.57 0.017 46 0.01 0.01Ipratropium-Br <0.1 Inhalation NA aerosol with very low plasmaexposure * Intrinsic inhibition constant assessed in protein free HBSS.** Apparent inhibition constant measured in 100% human serum or HBSScontaining 4% bovine serum albumin (BSA).

Example 2

The following example relates to the identification of clinicallyrelevant inhibitors of OCT2-mediated oxaliplatin uptake.

The FDA has published guidance in predicting if a drug is likely to be atransporter inhibitor in clinics by using data attained in in vitromodels [4]. C_(max,u) denotes the maximum free, unbound plasmaconcentration of a drug. If the ratio between C_(max,u) and in vitroIC₅₀ assessed in protein-free buffer is <0.1, the drug is unlikely toinhibit the transporter in vivo. Thus, characteristics of the in vitromodel may be used to predict in vivo effectiveness.

Accordingly, qualified inhibitors identified using the in vitro methodsdescribed above were examined for a potential effect on OCT2-mediatedoxaliplatin cellular uptake clinically by using published clinicalpharmacokinetic data of these drugs. Using this data, seven compoundshad calculated C_(max,u)/IC₅₀ ratios that are >0.1 (Table 2). Thesedrugs are buflomedil, miconazole, erlotinib, propantheline, epinastine,pentamidine, dolutegravir, and cimetidine. Among them, pentamidine,buflomedil and erlotinib had the highest C_(max,u)/IC₅₀ ratios (>1),suggesting that at their maximal clinical plasma concentrations, thesedrugs may inhibit over 50% OCT2-mediated oxaliplatin cellular uptake invivo. However, a clinical dose of pentamidine can cause nephrotoxicityin nearly 25% of patients, hence it may be a less desirable OCT2inhibitor for reducing platinum drug toxicity.

For other drugs, their low (<1) C_(max,u)/IC₅₀ ratios suggest that thesedrugs may have to be administered at a dose that is much higher thantheir normal clinical dose in order to achieve >50% OCT2 mediatedoxaliplatin uptake in vivo. For example, Tiotropium and epinastine areophthalmic drugs that were shown as very potent OCT2 inhibitors with ameasured IC₅₀ of 0.12 μM and 0.19 μM respectively. However, theirsystemic/plasma levels are also very low (Table 2), therefore, whenadministered at their approved clinical dose, they are likely to haveminimal inhibitory effects on OCT2 activities in organs other than theeye. Alternatively, changing their formulation and/or route ofadministration may increase their plasma exposure, which may induceunexpected systemic toxicities not observed at low plasma exposure.

In addition, another method was used to identify compounds that canpotentially affect OCT2-mediated oxaliplatin cellular uptake clinically.Instead of C_(max,u)/IC₅₀ ratio, C_(max)/IC_(50,app) ratio was used,where C_(max) denotes the total plasma concentration of a drug andIC_(50,app) denotes the apparent half-maximal inhibition constantmeasured in vitro in 100% serum or assay buffer containing physiologicalconcentrations of serum binding proteins, such as albumin. This approachwas demonstrated to have more accurate prediction of in vivo transporterinhibition effects of highly protein bound (>90%) compounds [17].

Using C_(max)/IC_(50,app) ratio, it was further demonstrated thatbuflomedil and erlotinib are likely to be clinically relevant inhibitorsof OCT2-mediated oxaliplatin cellular uptake. Moreover, it wasdemonstrated that two highly serum protein bound drugs miconazole anddolutegravir, which had C_(max,u)/IC₅₀ of less than 1 (Table 2), mayexhibit much higher inhibition of OCT2-mediated oxaliplatin cellularuptake in vivo because of their high C_(max).IC_(50,app) ratios (4.52and 3.23 respectively, Table 2).

In addition to looking at C_(max,u)/IC₅₀ or C_(max)/IC_(50,app) ratios,it is also important to factor in other pharmacokinetic (PK) parametersin order to find an inhibitor with optimal clinical effects wheninhibiting OCT2 mediated oxaliplatin uptake.

TABLE 3 Clinical pharmacokinetic parameters of buflomedil, miconazole,erlotinib, dolutegravir, and pentamidine Mean Mean Drug and typicalC_(max) C_(max) Mean T_(1/2) C_(max)/IC_(50, app) clinical dose (ug/mL)(uM) (hour) Reference ratio Buflomedil 2.1 6.8 3.4  [9] 4.69 (300 mg po,bid) Buflomedial 2.2 7.2 >10  [9] 4.97 (600 mg slow- release, po, qd)Miconazole 5 1.2 0.4 (distribution [7, 8] 3.64 (800 mg IV) phase), 8.9(terminal phase) Erlotinib (150 mg 1.14 2.9 2.0 [10] 1.39 po, singledose) Erlotinib (150 mg 2.12 5.4 18.2 [10] 2.60 po, day 24) Dolutegravir4.56 10.9 14.2 [15] 3.23 (50 mg po qd, single dose) Dolutegravir 6.1614.7 ~1.5 [15] 4.36 (50 mg po qd, day 10)

In clinical oncology, oxaliplatin is typically dosed through intravenousinfusion over a few hours. As shown in FIG. 1A, the plasma concentrationof oxaliplatin remains high within three hours after the start of thetwo hour infusion. As a drugs' efficacy and toxicity are typicallyclosely related to their plasma concentration, oxaliplatin may inducemore toxicity during this period. Therefore, to best reduce oxaliplatintoxicities that are be attributed to OCT2-mediated cellular uptake, aninhibitor of OCT2-mediated oxaliplatin uptake should maintain sufficientplasma concentration at least during this period. In the case ofmiconazole, 600 mg dosed IV bolus (FIG. 1B) has a high maximal plasmaconcentration; however, its plasma concentration drops rapidly with ahalf-time of 0.4 hour (initial phase), resulting in nearly five-folddecrease at 3 hour. Such a PK profile with short half-time may not beideal for sustaining the inhibition of OCT2-mediated oxaliplatin uptake(and thus inhibition of toxicity) in a clinical setting, unlessmiconazole is infused over time or the administration ofcisplatin/oxaliplatin is shorter. For instance, it is possible toadminister both cisplatin and oxaliplatin by bolus or by shorterinfusion time. In contrast to miconazole IV injection, buflomedil,dolutegravir and erlotinib oral administration have long half-times(Table 3), which means their plasma concentration can be maintained atdesirable concentrations for longer period of time (FIG. 1C, FIG. 1D andFIG. 1E). Buflomedil 600 mg slow-release tablet qd (FIG. 1C(2)) seems tobe particularly well adapted to protecting from 2 hour infusion ofplatinum derivatives, particularly for oxaliplatin infusion.

Example 3

The following example relates to the inhibition selectivity for OCT2over other major transporters involved in oxaliplatin handling.

In addition to OCT2, oxaliplatin is also thought to interact with otheruptake transporters, mainly OCT1 and OCT3. Efflux transporters includingMATE1 and MATE2K are also involved in controlling the cellularaccumulation of oxaliplatin. Applicants have confirmed thetransportability of oxaliplatin by transporters including OCT1, OCT2,OCT3 and MATE1 and MATE2K (FIG. 2).

These transporters have different roles in modulating oxaliplatinefficacy and toxicity. For example, OCT1 and OCT3 have been linked tooxaliplatin efficacy in tumor cells. However, MATE1 and MATE2K arebelieved to reduce oxaliplatin and cisplatin's toxicity in the kidney.Therefore, it is important to have a drug that specifically inhibitsOCT2 so that it will not affect oxaliplatin efficacy and/or aggravateoxaliplatin nephrotoxicity.

Using buflomedil as an example, the importance of selectivity ininteracting with oxaliplatin transporters was demonstrated. Erlotiniband cimetidine, two OCT2 inhibitors which have been tested in clinicaltrials for their ability to protect patients from platinum drug-inducedtoxicity, were used as comparison. Using a similar in vitro transportermethod as described above, the IC₅₀ of these drugs against OCT1, OCT2,OCT3, MATE1 and MATE2K was determined. Table 4 lists the measured orestimated serum apparent IC₅₀.p values for buflomedil, erlotinib,cimetidine, miconazole, and dolutegravir against oxaliplatin (20 μM in100% human serum or 100 μM in HBSS containing 4% BSA) transport mediatedby these transporters.

Buflomedil was demonstrated to be a selective inhibitor of OCT2 intransporting oxaliplatin, with at least 20-fold higher potency overOCT1, OCT3, MATE1 and MATE2K (FIG. 3A, Table 4). Dolutegravir was alsodemonstrated to be a potent selective inhibitor of OCT2 in transportingoxaliplatin, with at least 10-fold higher potency over OCT1, OCT3 andMATE1 (FIG. 3D, Table 4). Miconazole was demonstrated to be a potentinhibitor of OCT2 in transporting oxaliplatin, with at least 2-foldhigher potency over OCT1, OCT3, MATE1 and MATE2K (FIG. 3C, Table 4).Erlotinib was also demonstrated to be potent in suppressing OCT1 as wellas OCT2 (FIG. 3B, Table 4). Cimetidine was shown to be an inhibitor ofOCT2, but with even more preference towards inhibiting oxaliplatintransport through MATE1 (Table 3). These data suggested that compared toerlotinib and cimetidine, buflomedil, miconazole and dolutegravir are abetter agent, as they are more selective inhibitors of OCT2-mediatedoxaliplatin transport. Particularly, buflomedil and dolutegravir, whichshowed >10× selectivity between OCT2 and other relevant transporters(OCT1, OCT3, MATE1).

Compared to buflomedil and dolutegravir, miconazole has low (<2×)selectivity between OCT2 and OCT1, suggesting it is more likely tointerfere with oxaliplatin efficacy than the other two drugs in OCT1expressing tumors such as colon and liver cancers. Nevertheless, becausemicronazole has high selectivity (>10×) between OCT2 and other relevanttransporters (OCT3, MATE1 and MATE2K), it may still be a promisingcandidate when OCT1 play a minimal role in therapeutic efficacy (e.g.,when a chemo drug is an OCT2 substrate but not OCT1, and/or the tumorexpress low level of OCT1).

The same methods can be used to determine the transporter inhibitionselectivity of imidazole and other drugs.

Despite that buflomedil was discovered first by us to be a potent andselective inhibitor of OCT2 mediated oxaliplatin transport. FIG. 15Ashows that buflomedil had no inhibition selectivity between OCT2 andMATE1 when metformin was used as the substrate. The result furtherdemonstrates the critical importance of using oxaliplatin as thetransporter substrate in order to screen for the appropriate drugs forminimizing OCT2 mediated oxaliplatin toxicity.

In contrast, buflomedil was further evaluated for its OCT2 and MATE1inhibition potencies using cisplatin as the transporter substrate. FIG.15B shows that buflomedil has nearly identical IC₅₀s for OCT2 or MATE1mediated oxaliplatin and cisplatin transport. This result suggest thatcisplatin and oxaliplatin may interact with the same transporter bindingsites, leading to no or little substrate-dependent (oxaliplatin vs.cisplatin) buflomedil potency and selectivity as demonstrated.

TABLE 4 Apparent inhibition constants of oxaliplatin transport mediatedthrough different transporters OCT1 OCT2 OCT3 MATE1 MATE2K IC_(50, app)IC_(50, app) IC_(50, app) IC_(50, app) IC_(50, app) Compound (μM) (μM)(μM) (μM) (μM) Buflomedil 34.6 ± 7.6  1.4 ± 0.2 28.0 ± 11.4 32.7 ± 5.484.0 ± 23.1 Erlotinib 0.67 ± 0.06 2.1 ± 0.5 30.7 ± 4.9 no inhibition at10 μM Cimetidine 36.8 ± 13.5 Miconazole 8.4 ± 1.5 3.3 ± 0.6 49.2 ±22.6 >100 >100 Dolutegravir >100 3.4 ± 1.0 >100  42.7 ± 12.0Apparent inhibition constant, IC_(50,app) was determined in 100% humanserum or in HBSS containing 4% BSA, against transport of 20 μM or 100 μMoxaliplatin.

Example 4

The following example relates to the inhibition of intracellularoxaliplatin accumulation by buflomedil in cells co-expressing uptake andefflux transporters.

In most naturally occurring cells, uptake transporters co-exist withefflux transporters. It is the interplay between the uptake and effluxtransporters that determines the net intracellular accumulation of asubstrate. One example is in the proximal tubule epithelial cells in thekidney, where the basolaterally expressed transporters, mainly OCT2,work in concert with the apically expressed MATE1 and MATE2K [5]. Tomeasure how buflomedil can affect the net intracellular content ofoxaliplatin, a multi-transporter cell system was constructed byconcomitantly transfecting and expressing OCT2 and MATE1 at cDNA plasmidconcentrations of 40 and 10 ng/μL respectively. Cells were dosed withoxaliplatin 100 μM mixed with varying concentrations of buflomedil in 4%BSA HBSS for 90 min. At the end of the experiment, the intracellularcontent of oxaliplatin was quantified by LC/MS/MS. Cells with singleexpression of OCT2 or GFP mock were also used as a control.

As shown in FIG. 4, the intracellular content of OCT2-expressing cellswas significantly higher than cells expressing GFP mock control,suggesting OCT2 can potentiate oxaliplatin nephrotoxicity. However,cells expressing both MATE1 with OCT2 had significantly reduced cellularcontent of oxaliplatin in comparison with OCT2 only, suggesting thatMATE1 plays a role in protecting renal tubular cells from oxaliplatinassault. Because both buflomedil and erlotinib are more potentinhibitors of OCT2 than MATE1 (Table 4), they reduced cellularaccumulation of oxaliplatin in cells co-expressing OCT2 and MATE1,suggesting that they should also potentially reduce nephrotoxicityinduced by oxaliplatin. This is in contrast to cimetidine, a more potentinhibitor of MATE1 than OCT2, which may increase oxaliplatinaccumulation in renal tubular cells through preferential inhibition ofMATE1, hence potentially aggravating oxaliplatin induced nephrotoxicity.

Example 5

The following example relates to the discovery that buflomedil reducescytotoxicity of oxaliplatin by inhibiting its OCT2-mediated uptake.

Previous studies demonstrated that buflomedil is a potent inhibitor ofOCT2-mediated uptake of oxaliplatin. Here, reduction of oxaliplatincytotoxicity with buflomedil was tested with an in vitro MDCK (canineproximal epithelia cells) model overexpressing OCT2, MATE1 and MATE2K.

Materials and Methods

A triple-transporter model for OCT2(SLC22A2), MATE1(SLC47A1), andMATE2-K(SLC47A2) was created by transfecting fully confluent MDCK-IIcell monolayers with DNA mixture containing the plasmids encoding OCT2,MATE1, and MATE-2K at concentrations of 40, 15, and 5 ng/μl,respectively. The OCT2 was expressed on the basolateral cell membrane,and MATE1 and MATE2-K were expressed on the apical side of the cells. Todemonstrate the effects of MATE1/MATE2K efflux transporters, a modelexpressing similar level of OCT2 was also used. This systems werecultured in complete medium consisting of Dulbecco's modified Eagle'smedium without phenyl-red with 10% fetal bovine serum in an atmosphereof 5% CO2-95% air at 37° C. The apical medium was adjusted to pH 6.7with 25 mM of 2-(N-morpholino) ethanesulfonic acid (MES).

Both basolateral and apical sides were treated with variousconcentrations of buflomedil ranging from 3-1000 μM during thetreatment, and oxaliplatin was only added in basolateral side and fixedat 100 μM. Cells were pre-incubated with or without inhibitors for 30min, and then co-administered with oxaliplatin for further 4 hours.After removal of the media, a drug-free medium was added in both sides.After incubation 24 hours, the medium was collected, and the lactatedehydrogenase (LDH) activity in the medium was measured using an LDHcytotoxicity assay kit (G-Biosciences, St. Louis, Mo.; or Biochain,Newark, Calif.), according to the manufacturers' instructions.Cytotoxicity was evaluated by measuring the LDH activity in the medium.

Further, the concentration of an OCT2 inhibitor was fixed at aconcentration that is close to the drug's clinical C_(max), andco-administered with various concentrations of oxaliplatin ranging from1-1000 μM. Buflomedil was tested 3p and 6 μM. Dolutegravir was tested at10 μM. One competitor, erlotinib, was selected due to its great potencyon the inhibition of OCT2 uptake of oxaliplatin in previous studies.Another competitor, cimetidine, was also tested because it is a widelyknown non-selective OCT2 inhibitor, and because it has been studied bothin vitro and in vivo for reducing platinum induced toxicities, includingnephrotoxicity and neurotoxicity. Cytotoxicity was tested using the sameprocedure described above. In addition, cisplatin was reported as asubstrate of OCT2 transporter. Therefore, cisplatin was also tested at100, 300 and 1000 μM along with or without 6 μM of buflomedil in thistriple model. In another similar experiment, oxaliplatin cytotoxicitywas evaluated in OCT2 expressing cells with and without MATE1 and MATE2Ktransporters, to demonstrate the protective role of these effluxtransporters.

Results and Discussion

FIGS. 5 and 6 show that buflomedil and dolutegravir effectively reducedoxaliplatin-induced cytotoxicity in cells expressing OCT2. When 100 μMoxaliplatin was applied with buflomedil at different concentrations,buflomedil reduced oxaliplatin-induced cytotoxicity in a dose-dependentmanner, with a clinically relevant IC₅₀ value of 6 μM (FIG. 5).

To determine oxaliplatin cytotoxicity EC₅₀ values, cells were treatedwith various concentrations of oxaliplatin, in the presence and absenceof buflomedil, buflomedil, cimetidine or erlotinib at a fixedconcentration. FIG. 6A-B shows that compared to oxaliplatin treatmentalone, co-treatment with 3 μM and 6 μM buflomedil drastically reducedoxaliplatin cytotoxicity in cells expressing OCT2, MATE1 and MATE2K,with EC₅₀ values increased by 4.5× and 10× respectively, suggesting thatbuflomedil at clinically relevant concentrations can significantlyalleviate oxaliplatin-induced toxicity in OCT2 expressing cells.Similarly, 10 μM dolutegravir drastically reduced oxaliplatincytotoxicity in cells expressing OCT2, with and without MATE1 and MATE2K(FIG. 6C-D), with EC₅₀ values increased by 17.3× and 27.4× respectively.

Erlotinib showed cytotoxicity itself without adding oxaliplatin (FIG.6B). Although a reduction in toxicity of high concentration oxaliplatin(>100 μM) was observed in the presence of 6 μM erlotinib, its EC₅₀ valuecould not be obtained due to an ambiguous fitting. In contrast tobuflomedil and dolutegravir, 6 μM cimetidine did not reduce oxaliplatincytotoxicity in the triple transporter model, likely because it is astrong inhibitor of MATE1 and MATE2K but not OCT2. Overall, buflomediland dolutegravir showed superior effects compared to erlotinib andcimetidine in reduction of oxaliplatin cytotoxicity in theOCT2-expressing cells.

It should be noted that the triple-transporter model is particularlyuseful to model the disposition and toxicity of platinum drugs in renaltubular cells with abundant expression of OCT2 on the basolateral/plasmaside, and MATE1 and MATE2-K on the apical/tubular side. Hence, thesestudies particularly suggest that selective inhibitors of OCT2-mediatedoxaliplatin transport, such as buflomedil, can be used to reducenephrotoxicity associated with oxaliplatin.

In addition, FIG. 7 also suggests that buflomedil can reducecisplatin-induced cytotoxicity in cells expressing OCT2, MATE1 andMATE2K. The cisplatin-induced toxicity showed a dose-responserelationship after 30 μM treatment (data not shown). Threeconcentrations of cisplatin at 100, 300, and 1000 μM were compared.Compared to cisplatin alone, 6 μM buflomedil significantly reducedcytotoxicity of 1 mM cisplatin, suggesting buflomedil could be used toreduce cisplatin induced nephrotoxicity.

FIG. 16 shows MATE1 and MATE2K were effective on reducing oxaliplatincytotoxicity in OCT2 expressing cells. The presence of MATE1 and MATE2Knot only increased the EC50 of oxaliplatin cytotoxicity by 3×, but alsoreduced the maximum level of cell damage. These results were inagreement with the oxaliplatin accumulation study presented in Example 4and FIG. 4, further substantiating the hypothesis that MATE transportersplay an protective organs especially the kidney against toxicitiesinduced by oxaliplatin and cisplatin.

Example 6

The following example relates to the effects of buflomedil, dolutegravirand other drugs on in vitro anti-tumor effects of chemotherapeuticagents in tumor cell lines.

To evaluate whether buflomedil and dolutegravir could interfere withanti-tumor efficacy of oxaliplatin, cisplatin and chemotherapeutics usedalong with oxaliplatin, cytotoxicity assays were conducted in tumor celllines.

Materials and Methods

Anti-Proliferation Study in HT-29 and HepG2 Cells

HT-29 (a colorectal cancer cell line) and HepG2 (a hepatocarcinoma cellline) cells were selected to examine the potency of oxaliplatin whenco-administered with buflomedil, dolutegravir, cimetidine or erlotinib.The potency was determined by the sulforhodamine B (SRB) assay in96-well plates. HT-29 cells and HepG2 cells were maintained in McCOY's5A and Eagle's MEM media with 10% fetal bovine serum, respectively, inan atmosphere of 5% CO2-95% air at 37° C. Seeding density was 3000cells/well for both cell lines. After overnight incubation, 3 μM or 6 μMbuflomedil, 3 μM-9 μM dolutegavir, 3 μM or 6 μM of erlotinib, or 6 μM ofcimetidine were added to cells for pre-incubation for 30 min, and thenvarious concentrations of oxaliplatin ranging from 0.1 to 100 μM wereadded to the culture medium for further incubation of 72 hours.

Results and Discussion

Buflomedil, Dolutegravir and Cimetidine, not Erlotinib, Maintained thePotency of Oxaliplatin in HT-29 (Human Colorectal Adenocarcinoma) andHepG2 (Human Liver Carcinoma) Cells as Tested by Anti-ProliferativeStudies:

The anti-proliferative studies showed that 3 or 6 μM buflomedil, 3 μM or6 μM or 9 μM dolutegravir, did not affect oxaliplatin anti-proliferationIC₅₀ values compared to the treatment of oxaliplatin alone in HT-29(FIG. 8). In contrast, 6 μM erlotinib shifted oxaliplatin IC₅₀ by8.4-fold, suggesting that erlotinib may reduce the cytotoxicity ofoxaliplatin in HT-29 cells (FIG. 8A). A side-by-side comparison ofco-administration of oxaliplatin with buflomedil (6 μM), erlotinib (3μM), or cimetidine (6 μM) was also conducted. Only treatment oferlotinib with oxaliplatin significantly reduced the potency ofoxaliplatin (FIG. 9). In addition, the same results were obtained withHepG2 cells; the IC₅₀ value was shifted 10-fold when co-administeredwith erlotinib and oxaliplatin compared to treatment with oxaliplatinalone (FIG. 10). Without wishing to be bound by theory, it ishypothesized that both HT-29 and HepG2 cells express high levels ofOCT1. Therefore, oxaliplatin potency was reduced by erlotinib, which isa strong OCT1 inhibitor.

It is noteworthy that buflomedil and dolutegravir were shown to be astrong inhibitor of OCT2, but not OCT1 nor OCT3, in previous studies.Therefore, the potency of oxaliplatin remained the same when buflomedilor dolutegravir was co-administered. Although cimetidine does not reduceoxaliplatin potency when it was co-administered with oxaliplatin, it maynot effectively reduce cytotoxicity mediated by OCT2 due to its weakinhibitory effect on OCT2. Overall, the data suggested that buflomediland dolutegravir did not affect the anti-proliferation potency ofoxaliplatin on HT-29 and HepG2 cells due to weak or ignorable inhibitionof OCT1 or OCT3 in these cancer cells.

Cisplatin has also been reported as a substrate of OCTs. Therefore itwas tested whether these inhibitors reduce the potency of cisplatin inHT-29. None of the three inhibitors reduced cisplatin anti-proliferationpotency in HT-29 cells (FIG. 11). Interestingly, it was previously shownthat erlotinib can reduce the potency of oxaliplatin, possibly byinhibiting OCT1-mediated oxaliplatin uptake in tumor cells. Theseresults suggested that OCT1-mediated cisplatin transport may not be animportant mechanism of cisplatin accumulation in HT-29 cells. Thus,erlotinib has ignorable impact on cisplatin cytotoxicity in HT-29 cells.

In addition to oxaliplatin and cisplatin, buflomedil was tested for itspotential interference with 5-FU and gemcitabine, two chemotherapeuticscommonly used in combination with oxaliplatin in treating various tumorsincluding colorectal cancer and hepatocellular carcinoma. Again, asillustrated in FIGS. 12 and 13, buflomedil and dolutegravir did notimpact the anti-tumor effect of 5-FU and gemcitabine in HT-29 cells,suggesting co-administration of buflomedil and dolutegravir should notreduce efficacies of 5-FU and gemcitabine.

Example 7

The following example relates to platinum-induced neuropathy.

General methodologies known to the art are used to assess the activityof buflomedil following induction of various toxicities by platinumdrugs in selected neuropathy mice models. Other methods are available.Modifications of these methods are available as well.

Materials and Methods

Evaluation of General Toxicity

Mouse body weight is determined at baseline, before each drugadministration and every week up to 8 weeks. In addition, mice are alsoexamined daily for evaluation of general health including observationfor signs of hair loss, piloerection, general gait weakness, conditionof the hind paws and tail skin, and gastrointestinal disorders.

Ear cavity temperature is measured using an infrared thermometer (modelIRT303HACCP, National Product, MD) at baseline and after weeks 1, 3, 6,and 8 prior to performing the behavioral tests. Core body temperature ismeasured using a rectal probe (Thermalert TH-5 and TCAT-1A Controller,Physitemp Instruments, Inc.) at baseline and after weeks 1, 3, and 6 intwo mice from each drug treatment group after brief anesthesia withisoflurane or equivalent.

The nephrotoxicity of cisplatin and oxaliplatin is assessed by bloodurea nitrogen (BUN) levels in samples collected at the end of the 3-weekdrug treatment. Based on normal mouse BUN values (8-33 mg/dL accordingto normal reference laboratory values from Research Animal Resources atthe University of Minnesota-http://www.ahc.umn.edu/rar/refvalues.html;values as reported for normal untreated C57BL/6 mice), BUN levels >40mg/dL were used as an indication of developing nephrotoxicity.

Behavioral Testing

The order of testing is designed to ensure that the least stressful testis done first and to minimize the influence of one test on the nexttest. After a 2-week acclimation, the mice are trained five times overone week with the specific training protocols to familiarize them withtesting procedures prior to entering the study. For the von Frey and pawradiant heat test, mice are allowed to run freely in the apparatus for20 min before beginning the test. For the tail immersion test, eachmouse is loosely wrapped in a partially rolled, moist paper towel forone min; the practice episode is repeated for 3-4 times. For the gripstrength test, each mouse is guided for 5 min to pull the trapeze of thegrip strength meter 3-4 times. No training is used for the cold plateapparatus. All behavioral tests are carried out in groups of 4-6experimental mice at baseline, after completion of each drug treatmentcycle at weeks 1 and 3, and follow up evaluation at weeks 6 and 8. Allbehavioral tests are conducted at room temperature (25° C.) and betweenthe hour of 0900 and 1600 by one experimenter who is blinded to the drugtreatment condition.

Activity Monitoring

Monitoring of locomotor activity is carried out at baseline, during drugtreatment at weeks 1, 2, 3, and after treatment at weeks 6, 8, and 10using VersaMax Animal Activity Monitors (AccuScan Model RXYZCM-16,Columbus, Ohio). The activities of six mice are simultaneously evaluatedin six individual open chambers. Mice are allowed to run freely for 5min prior to behavioral recording for 20 min in the open chamber made ofa Plexiglas box (42 42×30 cm) with wood chip bedding. The VersaMaxmonitor has infrared sensors located every 2.54 cm along the perimeter(16 infrared beams along each side) and 2.5 cm above the floor. Althoughthe VersaMax monitor collects information in 21 behavioral categories,distance traveled is collected in 1-min intervals, collapsed into 102-min blocks, averaged and presented as group means f SEM.

Grip Strength Test

Grip strength is measured using a grip strength meter (Stoelting, WoodDale, II) as previously described. The grip strength meter consists of aforce transducer with digital display and a metal plate with a trapeze.Each mouse is placed on the plate and is pulled by its tail withincreasing force until it is unable to grasp the trapeze and the grip isbroken. The instrument digitally captures and displays the peakpull-force achieved. Muscle strength is defined as the peak weight (g)indicated on the display. The value is determined individually as themean of three trials and presented as group mean±SEM.

Cold Plate Assay

Temperatures ranging from −5° C. to 4° C. at 1° C. intervals are used toexamine the threshold for cold hyperalgesia in mice. Greaterreproducibility is obtained when counting the number of paw lifts in adefined period. In preliminary dosing studies, oxaliplatin-treated micedeveloped significant cold hyperalgesia between −5° C. and −3° C. Atemperature sensor is placed directly on the surface of the metal plateto ensure accurate temperature reading (TECA, Chicago, Ill.). For eachcold testing session, mice are brought to the testing room and allowedto acclimate for 10 min prior to being individually placed onto the coldmetal surface enclosed within a clear plexiglass barrier of 8 cm W×14 cmD×14 cm H with a top cover. To ensure the accuracy of paw lift counting,each cold plate testing session is videotaped using a video camcorder(Sony DCR-PC1000) and the video is replayed in slow motion. The totalnumber of brisk lifts of either hind paw or jumping is counted as theresponse to cold hyperalgesia. Movements associated with locomotion aredistinct, involving coordinated movement of all four limbs and theseshould be excluded. Mice are only tested once on any given test day toavoid any possible anesthetic or tissue damage effects that could beproduced by repeated exposure to a cold surface. A maximum cut off timeof 5 min is used to prevent tissue damage. Three separate trials arecarried out on three separate days at base line and two separate trialsduring and after drug treatment at weeks 1, 3, 6, and 8 are averaged andpresented as the mean number of paw lifts.

Von Frey Filament Assay

For the assessment of mechanical allodynia, an Ugo Basile DynamicPlantar Aesthesiometer (Stoelting, Wood Dale, II) using the von Freyfilament principle is used. Mice are placed under clear plastic boxesabove a wire mesh floor that allowed full access to the paws.Acclimation and exploratory behavior are observed for up to two hoursuntil mice become calm and close to motionless. The operator then placesthe touch stimulator apparatus under each mouse's hind paw and positionsthe calibrated metal filament below the target area of the hind paw.After pressing the start key, an electrodynamic actuator of theapparatus lifts the metal filament (diameter of 0.5 mm). The filamenttouches the plantar surface and exerts a continuous vertical force of 0to 5 g over a 10 s interval until the hind paw withdraws and activates astop signal. The instrument automatically registers the weight intensitythreshold in g that triggers paw withdrawal. Each hind paw is testedalternately with an interval of 5 min for four trials. Paw movementassociated with locomotion or weight shifting is not counted aswithdrawal responses. Paw withdrawal threshold of eight trials from bothhind paws of each mouse are averaged and recorded as mean±SEM.

Radiant Heat Assay

For the assessment of thermal hyperalgesia, a Hargreaves' test [16] isconducted using a Plantar Ugo Basile apparatus (Stoelting, Wood Dale,II). Mice can move freely in this apparatus on an elevated glass surfacewith plastic boxes above as the top cover. Mice are given a two houracclimation period prior to testing until they become calm andmotionless. A calibrated infrared light source of high intensity isapplied perpendicular on the plantar surface of each mouse's hind paw.The rising temperature on the bottom of the hind paw causes the mouse tomove its paw; the change in paw position alters reflected light andstops the timer. Latency to paw withdrawal is automatically recorded foreach trial. If the mouse does not withdraw its hind paw within 15 s, thetesting trial terminates to prevent tissue damage and 15 s is recorded.Each hind paw is tested alternately with an interval of 5 min for fourtrials. Paw withdrawal latency of eight trials from both hind paws ofeach mouse is averaged and recorded as mean±SEM.

Tail Immersion Assay

For assessment of tail thermal hyperalgesia, a tail immersion test isconducted as previously described with modifications. For each testingsession, mice are brought to the testing room and each is individuallyacclimatized three to four times in a moist paper towel for a minuteduration without tail immersion. Next, each mouse is swiftly and gentlywrapped in a slightly moist paper towel and held in the investigator'shand with minimal restraint to allow the distal one-third of the tail tobe immersed in a water bath. The water bath is preset at 50.5°±0.5° C.and the temperature is verified with an independent extra temperaturesensor. Only when the mouse is calm and its tail is relaxed, is the nextstep of tail immersion pursued. If the mouse becomes agitated, it isunwrapped and the testing protocol restarted. Latency to vigorous tailflick is recorded during three trials separated by at least 30 min, andthree trials are averaged and presented as mean±SEM. Cutoff time is setat 20 s, after which mouse is removed regardless of behavioral response.

Use of Buflomedil to Prevent or Treat Neurotoxicity of Oxaliplatin inMice

How buflomedil will prevent or treat neurotoxicity in oxaliplatininduced peripheral neuropathy model of mice will be measured asdescribed above and using the complete methodology described in Ta et al2009 [18]).

Thermal sensitivity associated with a single IP dose of oxaliplatin (40or 5 mg/kg) in male wild-type mice is assessed by a cold-plate test. Thenumber of paw lifts and licks when exposed to a temperature of −4, 4, or30° C. for 5 min is obtained for each mouse at 120 and 24 h beforereceiving oxaliplatin to determine the number of baseline events. Dataare recorded as the percentage change in the number of paw lifts or pawlicks compared with baseline values when the animals are exposed to thesame temperature 24 or 48 h after the administration of oxaliplatin.Mechanical allodynia is determined by a Von Frey Hairs test. Mice areleft to acclimate on a mesh platform and in 100×60 mm cylindrical tubesbefore the force necessary for a rigid Von Frey hair filament (IITC LifeScience) to induce paw withdrawal of the hind limbs is assessed for eachmouse at 120 and 24 h before receiving oxaliplatin to determine baselineevents. Paw withdrawal is assessed in triplicate on each hind paw with5-min intervals between repeating the test on each mouse and alternatingfrom the left paw to the right paw. Data are also recorded as thepercent change of force (in grams) necessary to promote paw withdrawalbefore and after the administration of oxaliplatin. In subsequentexperiments, the administration of oxaliplatin (5 mg/kg) is preceded byIV injection of buflomedil at several doses. Alternatively, buflomedilcan also be administered orally. In other experiments, buflomedil isactually administered 24 h before the administration of oxaliplatin,such that prevention properties could be observed.

Example 8

The following example relates to the use of buflomedil to prevent ortreat neurotoxicity of oxaliplatin in Oct1/2(−/−) mice.

Whether buflomedil will prevent or treat neurotoxicity in oxaliplatininduced peripheral neuropathy model of mice with both wild type andOct1/2(−/−) mice is measured using the materials and methods aredescribed above in Example 7 and the complete methodology is describedin [18].

Thermal sensitivity associated with a single IP dose of oxaliplatin (40or 5 mg/kg) in male wild-type mice and age-matched male Oct1/2(−/−) miceis assessed by a cold-plate test. The number of paw lifts and licks whenexposed to a temperature of −4, 4, or 30° C. for 5 min is obtained foreach mouse at 120 and 24 h before receiving oxaliplatin to determine thenumber of baseline events. Data are recorded as the percentage change inthe number of paw lifts or paw licks compared with baseline values whenthe animals are exposed to the same temperature 24 or 48 h after theadministration of oxaliplatin. Mechanical allodynia is determined by aVon Frey Hairs test. Mice are left to acclimate on a mesh platform andin 100×60 mm cylindrical tubes before the force necessary for a rigidVon Frey hair filament (IITC Life Science) to induce paw withdrawal ofthe hind limbs is assessed for each mouse at 120 and 24 h beforereceiving oxaliplatin to determine baseline events. Paw withdrawal isassessed in triplicate on each hind paw with 5-min intervals betweenrepeating the test on each mouse and alternating from the left paw tothe right paw. Data are also recorded as the percent change of force (ingrams) necessary to promote paw withdrawal before and after theadministration of oxaliplatin. In subsequent experiments, theadministration of oxaliplatin (5 mg/kg) is preceded by IV injection ofbuflomedil at several doses. Alternatively, buflomedil can also beadministered orally. In other experiments, buflomedil is actuallyadministered 24 h before the administration of oxaliplatin, such thatprevention properties can be observed.

Example 9

The following example relates to the use of buflomedil to protectagainst cisplatin nephrotoxicity.

Male wistar rats are treated systemically (IV) with PBS, cisplatin (11mg/kg), buflomedil (dose to be determined)+Cisplatin (11 mg/kg) orbuflomedil (5.5 mg/kg) alone. 72 hrs post treatment blood is collectedand analyzed for serum BUN (blood urea nitrogen) and serum creatininevalues.

It is expected that serum BUN values will increase with cisplatintreatment, while co-administration of buflomedil with cisplatin lowersthe BUN values. Serum creatinine values should also increasesignificantly with cisplatin treatment, however buflomedilco-administration with cisplatin should result in lower creatininelevels. Buflomedil treatment alone should not change the serumcreatinine values significantly when compared to control PBS-treatedvalues.

Example 10

The following example relates to the use of buflomedil to preventototoxicity for both oxaliplatin and cisplatin in trans-tympanic ratmodels.

Platinum drugs cause ototoxicity. Auditory brainstem responses (ABRs)have been measured prior to cisplatin or oxaliplatin administration(pretreatment ABRs), and 72 hours following cisplatin administration(post-treatment ABRs). A bar graph of auditory brainstem responses (ABR)threshold shifts (dB) observed under the different treatment conditionsindicating that cisplatin induces hearing loss, as evidenced by 20-35 dBshifts in thresholds, has been shown by others. It is expected thatthese threshold shifts could be abolished by buflomedil treatment at allfrequencies tested. Treatment with buflomedil alone should not affectthe ABR thresholds.

Buflomedil can be administered trans-tympanically to limit the concernthat it could interfere with the anticancer effects of platinum drugs.Trans-tympanic administration of drugs is the use of localizedapplication of drugs to prevent hearing loss. This route of drugadministration reduces the likelihood that the drug will get into thesystemic circulation and produce side effects or cause drug-druginteractions. The ease of drug delivery via the trans-tympanic routemeans that it can be readily performed on individuals in the out-patientsetting. The use of ventilation tubes in the tympanic membrane wouldallow for more episodic administration of buflomedil in children priorto administering chemotherapeutic regimen with oxaliplatin.

Pre-treatment ABRs are conducted on Wistar rats, which are thenpre-treated with vehicle or trans-tympanic buflomedil (dose of thesolution to be determined) followed by cisplatin (11 mg/kg, IP) or byoxaliplatin over a 30 min infusion period. Post-treatment ABRs areconducted 72 h later.

It is expected that trans-tympanic buflomedil will abolish cisplatin andoxaliplatin-induced hearing loss in rat model over all the threefrequencies tested in the vehicle-pretreated groups. Scanning electronphotomicrographs will provide a morphological analysis of the three rowsof outer hair cells of the organ of Corti to detect substantial outerhair cell damage. This damage should be abrogated by trans-tympanicadministration of buflomedil.

Example 11

The following example relates to a xenograft showing that both thereduction of nephrotoxicity of cisplatin and retention of cisplatinactivity in osteosarcoma cells is applicable to other cell linesincluding cell lines from colon and liver cancer.

Materials and Methods

Seven-week-old male Fischer rats and male Wistar rats are divided intofour groups (3 to 5 rats in a group): saline-injected control group(saline), buflomedil-alone group intravenously (IV) injected buflomedilvia a jugular vein by bolus injection (dose to be determined mg/kg) andcontinuous infusion using a microsyringe pump under light etheranesthesia (buflomedil alone), cisplatin-alone group intraperitoneally(IP) injected 1.75 mg/kg of cisplatin for tumor bearing rats or 7 mg/kgof cisplatin for rats without tumor (cisplatin alone) and combinedtreatment group injected cisplatin just before buflomedil injection(cisplatin buflomedil). The doses of cisplatin for tumor-bearing andun-bearing animals are the doses that show moderate antitumor effectagainst SOSN2 osteosarcoma and marked nephrotoxicity, based on theresults of the preliminary experiments. Similar studies can be conductedto determine what dose of cisplatin is required to have an effect onvarious cell lines including HT-29 for colon cancer and selected livercancer cell lines (see ref for full review [1]) for hepatocellularcarcinoma.

The animals are housed in a climate- and light-controlled environmentwith free access to water and food. Male Fischer rats are inoculatedwith a SOSN2 tumor block (about 10 mm3) subcutaneously on the back.Alternatively they are inoculated with colon or liver cancer cell lines.When tumor size reached about 300 mm³, drug treatments (cisplatin; 1.75mg/kg, IP, buflomedil according to the protocol) are performed. Thetumor size is measured every day for 16 d, as below.

Tumor size (mm³)=½×major axis×(short diameter)²

Male Wistar rats without tumors are treated with cisplatin (7 mg/kg) andbuflomedil (according to the protocol indicated), and blood samples (400ml) are collected from the tail vein under light anesthesia every day;the rats are killed by cervical dislocation under deep ether anesthesiaon day 5. Urine is collected for 24 h on the last day of the experiment.Creatinine and blood urine nitrogen (BUN) are measured in our laboratoryusing commercial kits, and other biochemical analyses are performed.

Measurement of Buflomedil Serum Concentration

One hundred microliters of the serum sample, 25 ml of the internalstandard ranitidine (100 mg/ml), and 100 ml NaOH (5 N) are mixed andbuflomedil is extracted with 3 ml methylene chloride, dissolved with 100ml of the mobile phase (5% acetonitrile/0.002 M triethylamine and 0.025%acetic acid), and measured using high-performance liquid chromatographyat 228 nm. Cells are cultured in RPMI-1640 medium (Sigma) supplementedwith 10%/o fetal bovine serum (Sigma) and 600 mg/ml kanamycin sulfate(Meiji Seika Co., Tokyo, Japan) in an atmosphere of 5% CO2 at 37° C.Cells (2.5-5103 cells/100 ml/well) are seeded onto 96-well plates, andafter 24 h, treated with varying concentrations of cisplatin in thepresence or absence of buflomedil (0.5, 1 mM) or NAC (3 mM) for 48 h.After treatment, cell viability is measured using the Cell CountingKit-8, based on the reduction activity of mitochondria dehydrogenases,according to the manufacturer's instructions.

Cellular Uptake of Cisplatin

For the measurement of cisplatin uptake, confluent growing cells in 100mm culture dishes are incubated with medium containing cisplatin (500mM) with or without buflomedil (1 mM) for 2 h. After treatment, thecells are rapidly washed twice with phosphate-buffered saline, thensolubilized by 0.5 N NaOH, and the protein content of the cell isdetermined with a Dc Protein Assay Kit (Bio-Rad Laboratories, Richmond,Calif., U.S.A.). Cell fluid solubilized with NaOH is diluted withdeionized Milli-Q water (Millipore, Billerica, Mass., U.S.A.) fivetimes. Elemental platinum concentrations are measured by flame atomicabsorption spectrometry on a model AA-6800 Atomic AbsorptionSpectrometer (Shimadzu Corporation, Kyoto, Japan). The absorbance ofatomized platinum is measured at 14 mA and a wavelength of 265.9 nm witha 0.5-nm slit width. Integrated absorbance with a read time of 5 s isrecorded. The standard curves are linear over a range of 0.5 to 50mg/ml. All measurements are performed in triplicate. The cellularplatinum levels are expressed as mg platinum per mg protein. Othertechnologies including mass spectrometry using platinum chelates couldbe used as well to measure the platinum concentrations.

In Vivo Experiments

In experiments described by others [14], serum concentrations ofcisplatin after IP injection (3.5 mg/kg) into rats resulted in a 24 hhalf-life, but cisplatin accumulated in the kidney for 48 h. Due to thisrapid clearance documented in rats, high serum concentrations ofbuflomedil after cisplatin injection should be maintained by an IV bolusinjection and continuous infusion for 4 h. The serum concentration ofbuflomedil can be maintained at adequate levels for 4 h using thisinjection protocol.

Initially, whether this buflomedil dosage influences the antitumoreffect of cisplatin will be examined. Typically, cisplatin significantlyinhibits tumor growth, and at 16 d after treatment, the tumor mass ofthe cisplatin-alone group is one-third that of the control group. Weexpect that buflomedil should have little influence on tumor growth inthe control group or in combination with cisplatin.

Next, a nephrotoxic dose of cisplatin (7 mg/kg, IP) is administered tonon-tumor rats, and the effect of buflomedil on the kidney function willbe examined. After administering cisplatin, kidney weight is typicallysignificantly elevated, and this increase should be restrained by theaddition of buflomedil, which when administered alone should not affectkidney weight. The serum creatinine and BUN levels should significantlyincrease after the third day of cisplatin injection, and buflomedilco-administration should significantly inhibit the increase of thesebiomarkers. As a result, rats are expected to suffer serious renaldamage 5 d after treatment with cisplatin, and the combined treatmentwith buflomedil should clearly reduce the damage.

In histopathology, necrotic and apoptotic changes in the epithelium cellof the renal tubule in the cisplatin treated group should be analyzedand the degree and range of the injury should decrease by thecombination of buflomedil.

Example 12

The following example relates to a assessing neurotoxicity intumor-bearing mice treated with oxaliplatin in the presence or absenceof buflomedil. A similar experiment could be performed with dolutegraviror miconazole or any other imidazole derivatives

The Use of Mice Models

Several studies have examined the neurophysiological, behavioral andpathological characteristics of oxaliplatin-induced peripheralneurotoxicity using rat models (Authier N, et al. Neurotherapeutics.2009; 6:620-629), and most of the oxaliplatin-induced pain studies havebeen done after a single injection of the drug. While rats developedsignificant cold and mechanical allodynia following a single dose ofoxaliplatin, these models are not representative of the chronicneurotoxicity experienced in clinical practice (Joseph E K, Levine J D.J Pain. 2009; 10:534-541). Cavaletti et al. (Eur J Cancer. 2001;37:2457-2463) have demonstrated that chronic oxaliplatin treatment inrats induced atrophy of dorsal root ganglia (DRG) neurons and decreasedperipheral sensory nerve conduction velocities (NCV). Moreover, chronicoxaliplatin treatment induced cold and heat hypersensitivity along withmechanical allodynia that persisted for 3 weeks after drug treatmentended (Ling B et al. Pain. 2007; 128:225-234). The use of rat models tostudy oxaliplatin-induced neurotoxicity has been very informative.However, since it is difficult to implant tumors in rats, most studiesof the anticancer properties of oxaliplatin have used mice. Thus, ratmodels have limited efficacy for investigations of peripheralneurotoxicity in the same experimental paradigms used to evaluate theanticancer activity of oxaliplatin.

The Use of Sophisticated Neuropathic Mice Models are More Representativeof the Pathology Observed in Humans

Recently, several mouse models of oxaliplatin-induced pain have beendeveloped using an acute, single dose or chronic, repeated doses ofoxaliplatin (Gauchan P et al. Neurosci Lett. 2009; 458:93-95). Whilethese studies demonstrated the development of mechanical and coldallodynia after oxaliplatin treatment, the characterization ofperipheral neurotoxicity was limited. To address these limitationsvarious studies performed in BALB/c mice treated with a schedule ofoxaliplatin were able to induce the onset of a painful neuropathy withthe aim to achieve a more complete characterization of the peripheraland central nervous system events induced by the chronic treatment(Renn, C. L. et al., Mol Pain, 2011, 7, 29).

The following experiments can be conducted in tumor-bearing mice both inthe presence and absence of buflomedil or other OCT-2 inhibitors withthe goal to reduce neurotoxicity while preserving anti-tumor propertiesof oxaliplatin.

General Appearance and Body Weight Change

To generate the model of oxaliplatin-induced painful peripheralneuropathy used in this study, the mice are given tail vein injectionsof oxaliplatin (3.5 mg/kg) twice weekly (separated by either 3 or 4days) for four weeks. The control group is naïve mice that do notreceive drug or vehicle injections. The duration of this study is 21days, preferably 30 days during which the mice are continuouslyallodynic after receiving oxaliplatin. The oxaliplatin is generallywell-tolerated by the mice. The mice are weighed on drug administrationdays and, over the course of the study, the oxaliplatin-treated micewill have a significant decrease in body weight compared to the naïvemice reaching about 15% by the completion of the study.

Neuropathological Analysis

Nerve conduction velocities (NCV) and action potential amplitude aremeasured in the caudal and digital nerves 4 days after the finaloxaliplatin dose. Chronic oxaliplatin treatment should induce asignificant decrease in the caudal NCV with a concomitant significantdecrease in action potential amplitude compared to the naïve group.

Morphological Analysis of DRG and Sciatic Nerve

To determine whether the altered function of peripheral neurons afteroxaliplatin are accompanied by structural changes in the DRG cell bodiesand axons of the sciatic nerve, thin sections through the L4-L5 DRGs andsciatic nerve from naïve and oxaliplatin-treated mice are examined atthe light and electron microscope levels two days after the final doseof drug in week four. Particular attention will be given to differencesbetween oxaliplatin treated and non-treated animals. Examination ofsciatic nerves by light microscope will allow clear observation of thestate of myelinated fibers in the sciatic nerve of oxaliplatin-treatedmice compared to naïve animals.

Previous work in rat models demonstrated that platinum-derived compoundsinduce DRG neuron cell body shrinkage (Carozzi V A et al, Exp Neurol.2010; 226:301-309). Since finding that oxaliplatin induced changes tothe nucleoli of DRG neurons in mice model, a morphometric analysis wasperformed to examine the cell bodies of DRG neurons fromoxaliplatin-treated (3.5 mg/kg/iv twice weekly for four weeks) and naïvecontrol mice for evidence of cell body shrinkage similar to that seen inrats. The morphometric analysis revealed that DRG neurons fromoxaliplatin-treated mice had a significant decrease in the area (mm2) oftheir cell bodies and nucleoli but not nuclei, compared to DRG neuronsfrom naïve mice (black bars). Morphological analyses are thereforerecommended both in presence and absence of OCT2 inhibitors likebuflomedil, dolutegravir and miconazole to demonstrate potentialprotection from oxaliplatin toxicity.

Reducing Neurotoxicity with Buflomedil Concurrent with Retention ofAnti-Tumor Activity in Tumor Bearing Mice Model Treated withOxaliplatin.

As shown in the previous examples, buflomedil clearly reduced theneurotoxicity induced by oxaliplatin in several mice models. The intentnow is to demonstrate that such reduction of neurotoxicity does notinterfere with the antitumor activity of oxaliplatin in both colon andliver xenografts mice models.

Four-week-old male BALB/c nude mice are used for that particularexperiment. Mice are housed under specific pathogen-free conditions;food and water are provided ad libitum. After the animals have been inquarantine for one week, they are implanted subcutaneously with a humancolorectal tumor cell line like HT-29 or HepG2 (human liver carcinoma)cells, the volume of which is approximately 8 mm3.

To evaluate the antitumor activity, the mice are grouped according tothe tumor volume once the mean tumor volume reached about 150 to 200 mm3(day 0). Each group consisted of 7 to 9 mice.

The tumor diameters are measured twice a week until day 21, and thetumor volume is estimated as 0.5×length×width2. The relative tumorvolume (RTV) is calculated using the following formula: RTV=(tumorvolume on measured day)/(tumor volume on day 0). On day 15, the tumorgrowth inhibition ratio (TGI) is calculated using the following formula:TGI=[1−(mean tumor volume of treated group)/(mean tumor volume ofcontrol group)]×100. The body weight change (BWC; %) is calculated as[(body weight on day 15)−(body weight on day 0)]/(body weight on day0)×100(%). The growth delay period (GDP), which indicates the differencein the period during which the RTV grew to 4 (corresponding to 50% ofthe size of the control tumors at the endpoint on day 21), is determinedaccording to a previously reported procedure (Balin-Gauthier D, et al,Cancer Chemother Pharmacol 7: 709-718, 2006)

Toxicity is defined as a 20% or more body weight loss or toxic death.Whether buflomedil will prevent or treat neurotoxicity inoxaliplatin-induced peripheral neuropathy model of mice with tumorbearing mice is measured using the materials and methods described abovein Examples 7 and 8. Neurotoxicity can also be measured by any of theexperimental methods described at the beginning of this example andcomprising morphological analysis of DRG and neurons, neuropathologicalanalysis, measure of body weight, and general appearances.

More specifically, neurotoxicity could be assessed by thermalsensitivity associated with a single IP dose of oxaliplatin (40 or 5mg/kg) in tumor bearing mice using a cold-plate test. The number of pawlifts and licks when exposed to a temperature of −4, 4, or 30° C. for 5min is obtained for each mouse at 120 and 24 h before receivingoxaliplatin to determine the number of baseline events. Data arerecorded as the percentage change in the number of paw lifts or pawlicks compared with baseline values when the animals are exposed to thesame temperature 24 or 48 h after the administration of oxaliplatin.Mechanical allodynia is determined by a Von Frey Hairs test. Mice areleft to acclimate on a mesh platform and in 100×60 mm cylindrical tubesbefore the force necessary for a rigid Von Frey hair filament (IITC LifeScience) to induce paw withdrawal of the hind limbs is assessed for eachmouse at 120 and 24 h before receiving oxaliplatin to determine baselineevents. Paw withdrawal is assessed in triplicate on each hind paw with5-min intervals between repeating the test on each mouse and alternatingfrom the left paw to the right paw. Data are also recorded as thepercent change of force (in grams) necessary to promote paw withdrawalbefore and after the administration of oxaliplatin. In subsequentexperiments, the administration of oxaliplatin (5 mg/kg) is preceded byIV injection of buflomedil at several doses. Alternatively, buflomedilcan also be administered orally. In other experiments, buflomedil isactually administered 24 h before the administration of oxaliplatin,such that prevention properties can be observed. The assessment ofneurotoxicity is performed in parallel with the assessment of tumorreduction, therefore, in addition to the measures performed prior toadding oxaliplatin, it is recommended to perform such assessment at 24,48, 72 hours and at day 7, 14 and 21. After initial treatment, the tumorvolume is measured twice a week continuously until day 21.

It is expected that administration of oxaliplatin will show significantantitumor activity against both colon and liver xenograft models. It isalso expected that several mice could die from oxaliplatin exposureindependently from the effect on neurotoxicity. Finally, it is expectedthat anti-tumor activity of oxaliplatin observed by measuring change intumor volume and body weight for both colon and liver tumor cellsmeasured in absence of buflomedil will be similar to the anti-tumoractivity of oxaliplatin in presence of buflomedil, preferentially withmeasurable reduction of neurotoxicity as defined in the examples aboveand in at the beginning of this example.

Example 13

The following example relates to the measurement of buflomedil plasmaconcentration in Balb/C mice via intravenous (IV) and intraperitoneal(IP) administration.

Experiments

Test article Buflomedil was prepared in 5% glucose formulation at 2mg/mL (for IV) or 4 mg/ml (for IP). Male mice of 7-8 weeks of age wereallowed to acclimate for 6 days. Three mice were administered 10 mg/kgof buflomedil via intravenous injection. Three mice were administered 20mg/kg, three mice were administered 40 mg/kg of buflomedilintraperitoneal injection. Blood was collected at 0.083, 0.5, and 1 hourinto Heparin tubes. The plasma was obtained through centrifugation ofthe blood at 4000 rpm at 4° C.

For plasma sample analysis, the samples were thawed and mixed wellbefore 20 μL aliquots were processed using protein precipitation byinternal standard (Verapamil) containing organic solution, methanol:acetonitrile 5:95 (v/v). After vigorous vortexing and refrigeratedcentrifugation at 4000 rpm, 50 μL of the extract from each sample wasreconstituted with 70 μL of 0.1% formic acid in water. Calibrationstandards were prepared by first serial diluting the 2 mg/mL Buflomedilstock solution with 70% acetonitrile and then spiking the solutions intoblank plasma. The calibration curve was extracted in the same way as theplasma samples. The final extracts were analyzed by LC-MS/MS usingpositive electrospray ionizations under the multiple-reaction-monitoring(MRM) mode for the detection of buflomedil. The standard curve fitted bylinear regression was used to quantify the analyte in the matrix usingAnalyst 1.5.2 software (AB Sciex)

Results

The bioanalytical method was successfully established to measurebuflomedil concentrations in mouse plasma. The lower limit ofquantitation (LLOQ) was 0.5 ng/mL. The buflomedil plasma levels arerepresented in FIG. 17A. The results show the average buflomedil plasmaconcentration in the mice of each group remained above 2 mg/l at 10minutes after IV/IP administration. The results further suggest that theestimated bioavailability of IP administration is between 40-60%.

Example 15

The following example relates to the measurement of dolutegravir plasmaconcentration in Balb/C mice via oral (PO), intravenous (IV) andintraperitoneal (IP) administration.

Experiments

Test article dolutegravir was prepared in DMSO:50 mM N-methylglucaminewith 3% Mannitol formulation (1:19 v/v) at 1 mg/mL. Male mice of 6-7weeks of age were obtained from Charles River and allowed to acclimatefor 3-5 days. The mice were weighed and individually identified (tailmarked). The mice (n=3) in the oral (PO) group were fasted overnight andorally gavaged with 10 mg/kg of Dolutegravir. The second group of mice(n=3) were intravenously (IV) injected with 4 mg/kg of Dolutegravir. Thethird group of mice (n=3) were intraperitoneally (IP) injected with 4mg/kg of Dolutegravir. At 10 min, 0.5 h, 1 h post dosing, ˜120 μl ofblood sample was collected from the IV and IP mice by orbital venoussinus bleeding. At 1 h, 2 h, 4 h post dosing, ˜120 μl of blood samplewas collected from the PO mice by orbital venous sinus bleeding. Theplasma was harvested by centrifugation of blood samples at 4000 rpm at4° C. for 10 minutes and stored in dry ice.

For analysis, the plasma samples were diluted into the calibration rangeas needed. Aliquot of 20 μL of each plasma sample was treated with 100μL of internal standard (Verapamil) containing organic solution,methanol: acetonitrile 5:95 (v/v). After vigorous vortexing andrefrigerated centrifugation at 4000 rpm, 50 μL of the supernatant fromeach sample was transferred to the injection plate and reconstitutedwith 70 μL of 0.1% formic acid in water. Calibration standards wereprepared by first serial diluting 2 mg/mL Dolutegravir stock solutionwith 70/6 acetonitrile and then spiking the solutions into blank plasma.The calibration curve was extracted in the same way as the unknownsamples. The final extracts were analyzed by LC-MS/MS using positiveelectrospray ionizations under the multiple-reaction-monitoring (MRM)mode for the detection of Dolutegravir. The standard curve fitted bylinear regression was used to quantify the analyte in the matrix usingAnalyst 1.6.2 software (AB Sciex).

Results

The dolutegravir plasma levels are represented in FIG. 17B. The resultsdemonstrate that the route of administration significantly affecteddolutegravir plasma exposure in mice.

Example 14

The following example relates to assess the neuroprotective effects ofbuflomedil, administered via IV and IP injection, in Balb/c mice exposedto chronic treatment with Oxaliplatin.

Summary

At the beginning of the study, 48 male Balb/c mice were randomized into4 experimental groups: one group was left untreated (CTRL, n=12), onegroup was treated with Buflomedil (BFMD, n=12), one group was treatedwith Oxaliplatin (OXA, n=12), and one group was co-treated withOxaliplatin and Buflomedil (OXA+BFMD, n=12).

At baseline (before drug treatment) and at the end of treatment, caudalnerve conduction study (NCS), behavioral tests (Dynamic and Cold Plate),histopathology study were performed. Serum samples were collected at theend of treatment.

This study showed that oxaliplatin induced peripheral neuropathy in thegroup of mice repeatedly treated with oxaliplatin, as evidenced by DRGnerve injury (FIG. 19-20), sciatic nerve injury (FIG. 21), mechanicalallodynia (FIG. 22), cold allodynia (FIG. 23), and reduced caudal nerveaction potential (FIG. 24) and nerve conduction velocity (FIG. 25).Buflomedil was effective on reducing OXAIPN in the OXA+BFMD group,compared with the OXA only group. Particularly, morphology examinationand morphometric assessment of DRG nerves showed that BFMD nearlycompletely prevented OXA's induced DRG neuronal injury, which is thehallmark of OXAIPN. Behavioral and NCS tests also demonstrated thatBuflomedil was effective on reducing OXAIPN.

Plasma level analysis of Buflomedil and Oxaliplatin confirmed thatBuflomedil was at an effective level that was expected to significantlyinhibit OCT2 mediated oxaliplatin transport, and oxaliplatin plasmalevel was not affected by buflomedil. All these results suggested thatin this study, buflomedil might be effective on abolishing OCT2 mediatedoxaliplatin accumulation in DRG, thus alleviating both the acute (coldallodynia) and chronic (mechanical allodynia and NCS) forms of OXAIPNthrough minimizing neural damage at least in DRG.

Study Aim

Buflomedil was discovered by us as a potent and specific inhibitor ofOCT2-mediated OXA transport (EXAMPLE 1); for this reason the purpose ofthis study was to verify if the inhibition of OCT2-mediated OXAtransport by Buflomedil is able to reduce the severity of OXA-inducedneuropathy in an established OXAIPN model (Renn C_(L)., Mol Pain, 2011,7, 29).

Materials and Methods

Animals were subjected to a physical examination (health check) shortlyafter arrival.

The care and husbandry of animals were in conformity with theinstitutional guidelines in compliance with national (D.L. n. 26/2014)and international laws and policies (EEC Council Directive 86/609, OJ L358, 1, Dec. 12, 1987; Guide for the Care and Use of Laboratory Animals,U.S. National Research Council, 1996).

Study Design

The study designed is represented in FIG. 18

Overall Experimental Plan

Treatment groups Animal numbers Control untreated animals (OXA)  1-12Buflomedil 30 mg/kg, ip + 15 mg/kg, iv (BFMD) 13-24 Oxaliplatin 3.5mk/kg (OXA) 25-36 Oxaliplatin 3.5 mk/kg + BFMD 30 mg/kg, 37-48 ip + 15mg/kg, iv (OXA + BFMD)

OXA was administered via the tail vein (iv). Intraperitoneal Buflomedil(ip) was injected 15 min before the co-administration of OXA and BFMDintravenously (iv)

Dose Selection Justification

Dosage and route of administration of Buflomedil were determined, basedon the data obtained from EXAMPLE 13, to achieve a target buflomedialplasma concentration greater than 1.72 mg/l during the course ofoxaliplatin administration. At such concentration, buflomedil isestimated to achieve >90% OCT2 inhibition based on in vitro data fromEXAMPLE 1. Dosage of Oxaliplatin was chosen based on previous experienceand the literature data (Renn C L., Mol Pain, 2011, 7, 29).

Study Schedule

Major activities Study day/week/month, time point Mortality DailyClinical signs Daily Body weight Two times a week Tissue sampling At theend of 4^(th) weeks of treatment Behavioral testing; Dynamic, Atbaseline and after 4^(th) weeks from Plantar and Cold Plate Test thebeginning of the study Neurophysiology After 4^(th) weeks from thebeginning of the study Pharmacokinetic analysis After 1^(st) and 8^(th)administration of OXA and BFMD Euthanasia CO₂ inhalation at sacrifice

Test Methods Nerve Conduction Velocity

The development of peripheral neuropathy after chronic administration ofdrugs was assessed by evaluating the nerve conduction velocity (NCV)along caudal and digital nerve using an electromyography apparatus(Myto2 ABN Neuro, Firenze, Italy). Caudal NCV was determined by placinga couple of recording needle electrodes at the base of the tail and acouple of stimulating needle electrodes 3.5 cm distally to the recordingpoints. Similarly, the digital NCV was determined by placing therecording electrodes close to the ankle bone and the stimulatingelectrodes close to the fourth toe near the digital nerve. Theintensity, duration and frequency of stimulation were set up in order toobtain optimal results. All the neurophysiological determinations wereperformed under standard conditions in a temperature-controlled room(22+/−2° C.) and the animal under isoflurane anesthesia along the wholeprocedure with continuous monitoring of vital signs.

Behavioral Test: Dynamic Plantar Aesthesiometer Test

The Dynamic Plantar Aesthesiometer Test was used to determine thealterations in mechanical pain perception and their changes due topharmacological treatment. Mice were accustomed to the device 3 daysbefore performing the tests after a 1 hour acclimatization periodfollowed by testing. The mechanical nociceptive threshold was assessedusing a Dynamic Aesthesiometer Test (model 37450, Ugo Basile BiologicalInstruments, Comerio, Italy), which generated a linearly increasingmechanical force. At each time point, after the acclimatization period,a servo-controlled mechanical stimulus (a pointed metallic filament,0.5-mm diameter) was applied to the plantar surface of the hind paw,which exerted a progressively increasing punctuate pressure, reaching upto 15 g within 15 seconds. The pressure evoking a clear voluntaryhind-paw withdrawal response was recorded automatically and taken asrepresenting the mechanical nociceptive threshold index. The mechanicalthreshold was always assessed alternatively on each side every 2 minuteson 3 occasions to yield a mean value. The results represent the maximalpressure (expressed in grams) tolerated by the animals. There was anupper limit cutoff of 30 seconds, after which the mechanical stimuluswas automatically terminated.

Behavioral Test: Cold Plate Test

Cold Plate Test was used to determine the alterations in coldtemperature related pain perception and their changes due topharmacological treatment. Cold Plate Test was performed by using anapparatus (35100—Hot/Cold Plate, Ugo Basile instruments) composed by aPlexiglas cylinder and a thermostatic plate, able to reach variabletemperatures. The mice were placed on the plate set at +4° C., free tomove and walk. Two blinded experimenters simultaneously determine thenumber of pain signs (e.g. jumping, licking, etc.) in a trial of 5minutes.

Neuropathological Analysis of DRG and Sciatic Nerve

The investigator was blind to the nerve and DRG conditions for theseanalyses. Three animals from each group were sacrificed four days afterthe last oxaliplatin dose. The left sciatic nerves and the L4-L5 DRGswere harvested and processed following previously reported protocols,resin embedded and sectioned for light and electron microscope analysis.For the light microscopy analysis, 1 μm semithin sections were stainedwith toluidine blue and examined using a Nikon Eclipse E200 lightmicroscope (Nikon, Firenze, Italy). For the electron microscopyanalysis, ultrathin sections (80 nm) counterstained with uranyl acetateand lead citrate, were examined with a Philips CM 10 transmissionelectron microscope (Philips, Eindhoven, Netherlands).

Morphometric Analysis of DRG Neurons

The investigator was blind to the experimental conditions of the tissuefor these experiments. One micron thick semithin sections stained withtoluidine blue were used for morphometric examination of the DRGs fromcontrol (n=3) and oxaliplatin-treated mice (n=3). (Renn et al. MolecularPain 2011, 7:29). Only the cells where the nucleolus was included in thesection plane of 50 μm-spaced out sections were considered to avoid theoverlapping of the same cell bodies and they were analyzed withcomputer-assisted image analysis (ImageJ, NIH) to measure the soma andnucleolus size of at least 300 DRG neurons per mouse.

Analysis of Sciatic Nerve Histology: Measure of G-Ratio

After completion of behavioral and electrophysiological analysis,animals were killed and sciatic nerves were dissected, fixed, stainedfor myelin and cut as semithin sections. Light microscopy was performedto capture an image of the entire nerve which was then subjected tosemi-automatic software-based nerve morphometry in order to assess axondiameter and myelin thickness 20.

Statistical Evaluation

The differences in body weight, NCV, sensory potential amplitude andbehavioral tests were statistically evaluated using the analysis ofvariance (ANOVA) and the Tukey-Kramer post-test (significance level setat p<0.05).

Data Acquisition

Data for all investigations were recorded, whenever possible, on-lineusing software packages specifically designed for the purposes of thetest facility/site. Where on-line recording was not possible,handwritten raw data sheets were used. The data were subsequentlyentered manually into the computer and the raw data sheets are archived.

Results Mortality

Two animals (BFMD and OXA+BFMD groups) died after the firstadministration; one animal (OXA+BFMD group) died after the lastadministration.

Clinical Observations

The administration of the test compounds was well-tolerated.

Body Weight

Body weight changes along the study. During the treatment all groups oftreatment did not show significant difference in body weight if comparedto untreated animals.

Morphometry and Histopathology Analysis

The morphometric analysis revealed that DRG neurons fromoxaliplatin-treated mice (OXA, dark grey) had a significant decrease inthe area (μm²) of their cell bodies (FIG. 19A) and nucleoli (FIG. 19B),compared to DRG neurons from naïve mice (CTRL, pale grey bars).Furthermore the presence of BFMD clearly protected the DRG with bothsomatic and nucleoli showing no difference from the CTRL group, after 4weeks of treatment with OXA (OXA+BFMD, black bars).

Thin sections through the L4-L5 DRGs and sciatic nerve from naïve,oxaliplatin-treated mice in presence or absence of BFMD treatment wereexamined at the light and electron microscope levels two days after thefinal dose of drug in week four (FIG. 20). Altered function ofperipheral neurons after oxaliplatin was accompanied by structuralchanges in the DRG cell bodies as it is clearly represented in (FIG. 20:OXA). Light microscopy revealed that DRG neurons fromoxaliplatin-treated mice had a high incidence of multinucleolated cellbodies with eccentric nucleoli compared to naïve DRG neurons. In allexperimental groups, the cytoplasm of neurons and satellite cellsappeared normal. BFMD groups in presence or absence of OXA look clearlysimilar to the control and therefore further establish the protectiveproperties of BFMD during treatment with OXA.

In summary, the presence of BFMD during treatment with OXA clearlyprotects DRG as observed both with cellular morphometry andhistopathology.

Neuropathological Analysis Sciatic Nerve

To assess changes in nerve cell myelination, we calculated the g ratioof nerve fibers (g-ratio denominates the numerical ratio between thediameter of the axon and the outer diameter of the myelinated fiber;hypomyelination in sciatic nerves, which is associated with platinumneurotoxicity (Boehmerle, W., et al. Sci. Rep. 4, 6370), would thuscause an increase of the g-ratio). As shown in FIG. 21, a mildhypomyelination, reflected as a small but statistically significantincrease in G-ratio, was evident in the OXA group as compared with CTRL.Treating with BFMD minimized such damage, showed as a reduction inG-ratio of the OXA+BFMD group vs OXA group. This data further confirmedthe protective effect of BFMD against nerve damage caused by OXA.

Dynamic Aesthesiometer Test

Dynamic Aesthesiometer Test results (FIG. 22) at baseline and at the endof the study are reported in Fig. O. At the end of treatment only thegroups treated with OXA alone showed the development of mechanicalallodynia with a reduction in the latency until withdrawal vs CTRL, BFMDand OXA+BFMD (p<0.001).

Cold Plate Test

Cold Plate Test results at baseline, after third injection and the endof the study (eight injections) are reported in FIG. 23. After thirdinjection only the group treated with OXA alone showed a significantincrease in pain signs versus CTRL and all groups of treatment(p<0.001). At the end of treatment both groups treated with OXA alone orin co-administration of BFMD showed a significant increase in pain signsversus CTRL and BFMD (p<0.001). The co-treated group showed asignificant decrease in pain signs versus OXA alone (p<0.05).

Neurophysiological Results

The administration of Oxaliplatin, induced a significant reduction incaudal nerve conduction velocity vs. control mice, the group treatedwith OXA+BFMD showed a reduction compared to CTRL and BFMD groups, butshowed a small but statistically significant increase when compared toOXA alone (FIG. 24). Only the OXA group showed a significant decrease incaudal amplitude (p<0.01 vs CTRL) (FIG. 25), whereas there was nostatistically significant difference among CTRL, BFMD and OXA+BFMDgroups.

Plasma Levels

Plasma levels for both OXA and BFMD were measured at the end of thestudy. Plasma levels for BFMD were consistently above an expected level(1.72 mg/l) (FIG. 26A). No pharmacokinetic interaction was observed invivo between BFMD and OXA as shown in FIG. 26A, whereby both levels ofBFMD and BFMD in presence of OXA are sensibly equivalent. Furthermoreplasma levels of OXA seems unaffected by the presence of BFMD (FIG. 26B)

Example 16

The following example relates to evaluating and comparison of the effectof Dolutegravir (DTG) and chlorphenesin (CTC) on acute peripheralneuropathy induced by a single administration of Oxaliplatin

Aim

The aim of this study is to assess the effects of Dolutegravir andChlorphenesin Carbamate in Balb/c mice exposed to acute treatment withOxaliplatin (OXA). Both dolutegravir and chlorphenesin are known OCT2inhibitors. However, in our experiments, we have previously established(EXAMPLE 1) that Dolutegravir is a selective OCT2 inhibitor whereasChlorphenesin does not inhibit OCT2 mediated Oxaliplatin transport,despite it was reported as a very potent inhibitor of OCT2 mediatedtransport of a non-platinum compound ASP+. Based on findings fromEXAMPLE 15, we expect Dolutegravir should block oxaliplatin uptake inDRG whereas Chlorphenesin should not. It is therefore our aim to observehow Dolutegravir and Chlorphenesis could differently protect male Balb/cmice in an acutely induced peripheral neuropathy

Study Design

At the beginning of the study, 30 male Balb/c mice were randomized (FIG.27) into 6 experimental groups: one group was left untreated (CTRL,n=5), one group was treated with Oxaliplatin iv 3.5 mg/kg (OXA, n=5),one group was treated with Dolutegravir 4 mg/kg ip (DTG, n=5), one groupwas treated with Chlorphenesin 4 mg/kg ip (CPC, n=5), one group wasco-treated with Oxaliplatin 3.5 mg/kg iv and Dolutegravir administeredip 60 min before the OXA administration (OXA+BFMD, n=12) and the lastgroup was treated with Chlorphenesin 4 mg/kg ip Oxaliplatin 3.5 mg/kgand Dolutegravir administered ip 60 min before the OXA administration(OXA+CPC).

The dose and route of administration of dolutegravir used in this studywere determined based on PK study in EXAMPLE 14, such that DTG plasmalevel should be sufficient to block OCT2 significantly (>90′%).

At baseline and at the end of treatment behavioral tests (Dynamic andCold Plate) and serum collection were performed. The behavioral testsevidenced the development of allodynia in the animals treated with OXAalone.

Drug Dosage and Formulation

Oxaliplatin (OXA), 3.5 mg/kg^(a), intravenously, dissolved in glucosate5%

Dolutegravi (DTG), 4 mg/kg^(b), intraperitoneally, formulated as asolution in DMSO diluted with 50 mM N-methylglucamine in 3% aqueousmannitol (1/19 v/v).

Chlorphenesin Carbamate (CPC), 4 mg/kg^(c), intravenously, dissolved inglucosate 5%

Pharmacological Treatment

OXA vehicle, OXA, CPC, DTG, DTG+OXA and CPC+OXA were administered oncetime. In the group co-administered with OXA and DTG, DTG was injectedintraperitoneally 1 hour before the administration of OXA. In the groupco-administered with OXA and CPC, CPC was co-administered with OXAintravenously. Mechanical allodynia and cold sensory threshold (dynamictest and cold plate test) were performed 24 and 48 hours after thetreatment, respectively, using the methods described in EXAMPLE 15

Plasma Sample Collection

15 min after the iv administration of oxaliplatin the serum sample werecollected for pharmacokinetic analysis from each group.

Results

FIG. 28A shows that a single injection of 3.5 mg/kg oxaliplatin inducedmechanical allodynia evidenced by a significant decrease in MechanicalPain Threshold (MPT) in the OXA group, as compared to the CTRL group, aswell as the DTG alone and OXA+DTG. There was no statisticallysignificant difference among the CTRL, DRG and OXA+DTG groups,suggesting DTG was effective in nearly completely alleviated mechanicalhypersensitivity acutely induced by oxaliplatin. In contrast, CPC wasshown to have no such effect as shown in FIG. 28B, which was an expectedresult for compounds that do not protect against OCT2 mediated platinumuptake in the DRG.

Similarly, cold plate assays results clearly show that DTG (FIG. 29)added to OXA protects the animal from cold allodynia.

These results clearly show that DTG, as expected, had a protectiveeffect on both types of allodynia and therefore could be used tominimize both acute and chronic side effects of CIPN and morespecifically OXA-IPN.

Example 16

The following example relates to correlating oxaliplatin accumulationwith OXAIPN in mice treated with eight cycles of oxaliplatin, with andwithout orally administered buflomedil

Study Design

This study had the same design as that described in EXAMPLE 15 exceptthat Buflomedil was administered by oral gavage (PO), 80 mg/kg, 2 hoursbefore oxaliplatin administration.

At the end of 8 cycles of treatment, cold plate test, dynamic test andNCS test were conducted in all mice. Platinum content in DRG, Sciaticnerves and kidney of 3 mice each in the OXA and OXA+BFMD groups, wasmeasured using the method below.

Platinum Accumulation Measurement

The tissue total platinum concentration was determined on frozen sciaticnerve, DRG, kidney, and plasma specimens collected from 3 animals/groupkilled 24 h after the last administration of the drugs. For each tissue,a calibration with control standard tissue was generated. All frozentest samples and standards were treated for a digestion process with aspecific HNO₃:HCl solution (1:3). The samples obtained after digestionwere analyzed by “Atomic Absorption” (Analyst 600 Perkin Elmer, Monza,Italy), and platinum tissue concentration was calculated accordingly.

Results

Similar to the results presented in EXAMPLE 15, orally administratedbuflomedil, at 80 mg/kg, showed protective effects against OXAIPN basedboth behavioral tests (mechanical pain threshold and cold plate test)(FIG. 30) and nerve conduction velocity measurement (FIG. 31). Moreover,buflomedil treatment resulted in a significant decrease in oxaliplatinaccumulation in DRG (FIG. 32A), whereas such decrease was not seen insciatic nerves that may not express high level of OCT2. The resultfurther support that buflomedil may protect oxaliplatin induced DRGdamage through reducing oxaliplatin accumulation via OCT2.Interestingly, buflomedil also reduced oxaliplatin accumulation in thekidney, suggesting in this study, buflomedil was able to block OCT2 inthe kidney. This result, together with ones shown in previous examples,further suggest that buflomedil and other selective OCT2 inhibitors,such as dolutegravir, are very likely to have a protective role againstcisplatin induced nephrotoxicity through reducing cisplatin accumulationin the kidney.

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1-109. (canceled)
 110. A method for reducing platinum drug-inducedtoxicity in a human subject in need thereof comprising: administering aplatinum drug that is cisplatin to the subject in need thereof; andadministering an effective dose of a selective Organic CationTransporter 2 (OCT2) inhibitor to the subject in need thereof, whereinthe OCT2 inhibitor is selected from the group consisting of buflomedil,a buflomedil salt, dolutegravir, and a dolutegravir salt. wherein thetoxicity is nephrotoxicity or ototoxicity.
 111. The method of claim 110,wherein the method is for increasing patient compliance for treatingcancer in the subject, whereby said subject completes treatment with acumulative dose of the platinum drug of at least 100 mg/m².
 112. Themethod of claim 110, wherein the selective OCT2 inhibitor results in itsplasma level during the period of platinum administration at least 0.43mg/L, 0.86 mg/L, 1.29 mg/L, 1.72 mg/L or 2.15 mg/L.
 113. The method ofclaim 110, wherein the selective OCT2 inhibitor does not reduce theefficacy of cisplatin.
 114. The method of claim 110, wherein the subjectin need has cancer, and wherein the cancer expresses at least one ofOrganic Cation Transporter 1 (OCT1) or Organic Cation Transporter 3(OCT3).
 115. The method of claim 114, wherein the cancer is selectedfrom the group consisting of colorectal cancer, liver cancer, gastriccarcinoma, pancreatic adenocarcinoma, esophageal or esophagogastricjunction carcinoma, lung cancer, head and neck cancer, and lymphoma.116. The method of claim 110, wherein the selective OCT2 inhibitor isadministered enterally, intravenously, intramuscularly,intraperitoneally, orally, or parenterally.
 117. The method of claim110, wherein the selective OCT2 inhibitor is buflomedil or a buflomedilsalt.
 118. The method of claim 110, wherein the selective OCT2 inhibitoris dolutegravir and a dolutegravir salt.
 119. The method of claim 110,wherein the subject in need thereof has liver cancer.
 120. The method ofclaim 110, wherein the subject in need thereof has gastric carcinoma.121. The method of claim 110, wherein the subject in need thereof haspancreatic adenocarcinoma.
 122. The method of claim 110, wherein thesubject in need thereof has esophagogastric junction carcinoma.
 123. Themethod of claim 110, wherein the subject in need thereof has lungcancer.
 124. The method of claim 110, wherein the subject in needthereof has head and neck cancer.
 125. The method of claim 110, whereinthe subject in need thereof has lymphoma.
 126. A method for reducingplatinum drug-induced nephrotoxicity in a human subject in need thereofcomprising: administering a platinum drug that is cisplatin to thesubject in need thereof; and administering an effective dose of aselective Organic Cation Transporter 2 (OCT2) inhibitor selected fromthe group consisting of buflomedil, a buflomedil salt, dolutegravir, anda dolutegravir salt to the subject in need thereof, wherein the subjectin need thereof has cancer, and wherein the cancer expresses at leastone of Organic Cation Transporter 1 (OCT1) and Organic CationTransporter 3 (OCT3)
 127. A method for reducing platinum drug-inducedototoxicity in a human subject in need thereof comprising: administeringa platinum drug that is cisplatin to the subject in need thereof; andadministering an effective dose of a selective Organic CationTransporter 2 (OCT2) inhibitor selected from the group consisting ofbuflomedil, a buflomedil salt, dolutegravir, and a dolutegravir salt tothe subject in need thereof, wherein the subject in need thereof hascancer, and wherein the cancer expresses at least one of Organic CationTransporter 1 (OCT1) and Organic Cation Transporter 3 (OCT3).
 128. Apharmaceutical composition for reducing platinum drug-inducednephrotoxicity or ototoxicity, formulated for intravenous administrationcomprising a platinum drug that is cisplatin, a selective OCT2 inhibitorselected from the group consisting of buflomedil, a buflomedil salt,dolutegravir, and a dolutegravir salt, and a pharmaceutically acceptablecarrier.
 129. A kit comprising a selective OCT2 inhibitor that isbuflomedil or a buflomedil salt, or dolutegravir or a dolutegravir salt;for use in reducing cisplatin-induced toxicity wherein the toxicity isnephrotoxicity or ototoxicity in a subject having cancer in a subject;and instructions for use.